Method of modulating stress-activated protein kinase system

ABSTRACT

Disclosed are methods of modulating a stress activated protein kinase (SAPK) system with an active compound, wherein the active compound exhibits low potency for inhibition of at least one p38 MAPK; and wherein the contacting is conducted at a SAPK-modulating concentration that is at a low percentage inhibitory concentration for inhibition of the at least one p38 MAPK by the compound. Also disclosed are derivatives of pirfenidone. These derivatives can modulate a stress activated protein kinase (SAPK) system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/679,471 filed on May 10, 2005, and U.S. ProvisionalPatent Application No. 60/732,230 filed on Nov. 1, 2005, both of whichare hereby expressly incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to compounds and methods useful in treatingvarious inflammatory conditions and/or fibrotic conditions, includingthose associated with enhanced activity of kinase p38.

BACKGROUND OF THE INVENTION

A large number of chronic and acute conditions have been recognized tobe associated with perturbation of the inflammatory response. A largenumber of cytokines participate in this response, including IL-1, IL-6,IL-8 and TNFα. It appears that the activity of these cytokines in theregulation of inflammation may be associated with the activation of anenzyme of the cell signaling pathway, a member of the MAP kinase familygenerally known as p38 and also known as SAPK, CSBP and RK.

Several inhibitors of p38, such as NPC 31169, SB239063, SB203580,FR-167653, and pirfenidone have been tested in vitro and/or in vivo andfound to be effective for modulating inflammatory responses.

There continues to be a need for safe and effective drugs to treatvarious inflammatory conditions and/or fibrotic conditions such asinflammatory pulmonary fibrosis and/or idiopathic pulmonary fibrosis.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method of modulating astress activated protein kinase (SAPK) system, including contacting acompound with a p38 mitogen-activated protein kinase (MAPK), wherein thecompound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μMfor inhibition of at least one p38 MAPK; and wherein the contacting isconducted at a SAPK-modulating concentration that is less than an EC₃₀for inhibition of the at least one p38 MAPK by the compound.

Another embodiment of the present invention is a method of treating orpreventing a disease state in a subject, including, identifying asubject at risk for or having a condition selected from an inflammatorycondition and a fibrotic condition; administering a compound to thesubject in an effective amount to treat or prevent the condition;wherein the compound exhibits an EC₅₀ in the range of about 1 μM toabout 1000 μM for inhibition of at least one p38 MAPK; and wherein theeffective amount produces a blood or serum or another bodily fluidconcentration that is less than an EC₃₀ for inhibition of the at leastone p38 MAPK.

Another embodiment of the present invention is a method of identifying apharmaceutically active compound, including: providing a library ofcompounds; assaying a plurality of compounds from the library forinhibition of at least one p38 MAPK; and selecting at least one compoundfrom the plurality of compounds, wherein the selected compound exhibitsan EC₅₀ in the range of about 1 μM to about 1000 μM for inhibition ofthe at least one p38 MAPK.

Another embodiment of the present invention is a method of identifying apharmaceutically active compound, including: providing a library ofcompounds; assaying a plurality of compounds from the library forinhibition of TNFα secretion in a bodily fluid in vivo; and selecting atleast one compound from the plurality of compounds, wherein the selectedcompound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μMfor inhibition of TNFα secretion in a bodily fluid in vivo.

Another embodiment of the present invention is a method of identifying apharmaceutically active compound, including: providing a library ofcompounds; assaying a plurality of compounds from the library forinhibition of TNFα secretion by cultured cells in vitro; and selectingat least one compound from the plurality of compounds, wherein theselected compound exhibits an EC₅₀ in the range of about 1 μM to about1000 μM for inhibition of TNFα secretion by cultured cell in vitro.

Another embodiment of the present invention is a method of identifying apharmaceutically active compound, including: providing a library ofcompounds; assaying a plurality of compounds from the library forinhibition of TNFα secretion in a bodily fluid in vivo; and selecting atleast one compound from the plurality of compounds, wherein the selectedcompound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μMfor inhibition of TNFα secretion by cultured cells in vitro.

Another embodiment of the present invention is a method of identifying apharmaceutically active compound, including: providing a library ofcompounds; assaying a plurality of compounds from the library forinhibition of TNFα secretion by cultured cells in vitro; and selectingat least one compound from the plurality of compounds, wherein theselected compound exhibits an EC₅₀ in the range of about 1 μM to about1000 μM for inhibition of TNFα secretion in a bodily fluid in vivo.

Another embodiment of the present invention is a compound having theformula of Subgenus III:

Wherein X₃ is selected from the group consisting of H, F, and OH; and R₂is selected from the group consisting of H and CF₃; and wherein thecompound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μMfor inhibition of p38 MAPK; or a pharmaceutically acceptable salt,ester, solvate or prodrug of the compound.

Another embodiment of the present invention is a compound having theformula of Genus VI:

Wherein Ar is pyridinyl or phenyl; Z is O or S; X₃ is H, F, Cl, OH, orOCH₃; R₂ is methyl, C(═O)H, C(═O)CH₃, C(═O)O-glucosyl, fluoromethyl,difluoromethyl, trifluoromethyl, methylmethoxyl, methylhydroxyl, orphenyl; and R₄ is H or hydroxyl; with the proviso that when R₂ istrifluoromethyl, Z is O, R₄ is H and Ar is phenyl, the phenyl is notsolely substituted at the 4′ position by H, F, or OH; wherein thecompound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μMfor inhibition of p38 MAPK; or a pharmaceutically acceptable salt,ester, solvate or prodrug of the compound.

Another embodiment of the present invention is a compound having theformula of Genus VII:

Wherein X₃ is H, halogen, C₁-C₁₀ alkoxy, or OH; Y₁, Y₂, Y₃, and Y₄ areindependently selected from the group consisting of H, C₁-C₁₀ alkyl,substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ haloalkyl, C₁-C₁₀nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ alkoxy,phenyl, substituted phenyl, halogen, hydroxyl, C₁-C₁₀ alkoxyalkyl,C₁-C₁₀ carboxy, C₁-C₁₀ alkoxycarbonyl; R₄ is H, halogen, or OH; andwherein the compound exhibits an EC₅₀ in the range of about 1 μM toabout 1000 μM for inhibition of p38 MAPK; or a pharmaceuticallyacceptable salt, ester, solvate or prodrug of the compound.

A further embodiment of the present invention is a pharmaceuticalcomposition containing a compound having a formula as described aboveand a pharmaceutically acceptable excipient.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the p38 MAPK signalling cascade(prior art, FIG. 1 from Underwood et al. 2001 Prog Respir Res31:342-345). This schematic shows activation of the p38 signalingcascade by a variety of stimuli and the downstream effects of p38activation on an inflammatory response through transcriptionalactivation and translation/mRNA stabilization.

FIG. 2 is a plot showing the fractional inhibition of p38γ by variouspirfenidone metabolites and analogs as a function of the concentrationof the compounds. The EC50 concentrations of these compounds are shownto the right of the plot. A detailed description of the assay can befound in Example 5.

FIG. 3 is a plot showing the fractional inhibition of p38α by variouspirfenidone metabolites and analogs as a function of the concentrationof the compounds. The EC₅₀ concentrations of these compounds are shownto the right of the plot. A detailed description of the assay can befound in Example 5.

FIG. 4 is a summary of the biochemical data for various pirfenidonemetabolites and analogs. The pirfenidone metabolites and analogsreferred to in FIGS. 2, 3, 5, and 6 are described by the substitutionpattern shown in FIG. 4. The summary shows the effect of substitutionsat three positions on the EC50 concentrations for inhibition of p38α andp38γ.

FIG. 5 is a plot showing the fractional activity (TNFα release frommacrophage in response to LPS) of various pirfenidone metabolites andanalogs as a function of the concentration of each compound. A detaileddescription of the assay can be found in Example 5.

FIG. 6 is a series of bar charts showing the cytotoxicity of variouspirfenidone metabolites and analogs at various concentrations of thecompounds. Cytotoxicity was determined by measuring LDH releasefollowing incubation of cells in the presence of the compound. Theamount of LDH released is normalized to that released upon treatmentwith Triton-X-100 and plotted versus compound concentration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has now been discovered that a high therapeutic effect in treatingvarious disorders associated with enhanced activity of kinase p38 may beachieved by using a relatively low-potency p38 kinase inhibitorcompound.

Therefore, in one embodiment there is provided a method of modulating astress-activated kinase (SAPK) system by contacting a compound with ap38 mitogen-activated protein kinase (MAPK). A preferred compoundexhibits an EC₅₀ in the range of about 1 μM to about 1000 μM, preferablyabout 50 μM to about 650 μM for the inhibition of at least one p38 MAPK.The concentration at which the compound is contacted with the p38 MAPKis generally less than EC₃₀ for inhibition of the p38 by this compound.Preferably, the concentration is less than EC₂₀, even more preferably,the concentration is less than EC₁₀.

“Mitogen-activated protein kinases (MAPKs)” are evolutionarily conservedserine/threonine kinases involved in the regulation of many cellularevents. Several MAPK groups have been identified in mammalian cells,including extracellular signal-regulated kinase (ERK), p38, andSAPK/JNK. It is believed that MAPKs are activated by their specific MAPKkinases (MAPKKs): ERK by MEK1 and MEK2, p38 by MKK3 and MKK6, andSAPK/JNK by SEK1 (also known as MKK4) and MKK7 (SEK2). These MAPKKs mayalso be activated by various MAPKK kinases (MAPKKKs) such as Raf, MLK,MEKK1, TAK1, and ASK1.

It is believed that the MAPK network involves at least twelve clonedhighly conserved, proline-directed serine-threonine kinases which, whenactivated by cell stresses (oxidative stress, DNA damage, heat orosmotic shock, ultraviolet irradiation, ischemia-reperfusion), exogenousagents (anisomycin, Na arsenite, lipopolysaccharide, LPS) orpro-inflammatory cytokines, TNF-α and IL-β, can phosphorylate andactivate other kinases or nuclear proteins such as transcription factorsin either the cytoplasm or the nucleus (FIG. 1).

p38 MAPK

As used herein, “p38 MAPK” is a member of the stress-activated proteinkinase family, which includes at least 4 isoforms (α, β, γ, δ), severalof which are considered important in processes critical to theinflammatory response and tissue remodeling (Lee et al. 2000Immunopharmacol. 47:185-201). The predominant kinases in monocytes andmacrophages, p38α and p38β, appear more widely expressed compared top38γ (skeletal muscle) or p38δ (testes, pancreas, prostate, smallintestine, and in salivary, pituitary and adrenal glands). The p38γisoform is expressed in myofibroblasts, which have some phenotypicsimilarities to muscle cells including expression of alpha-smooth muscleactin. A number of substrates of p38 MAP kinase have been identifiedincluding other kinases (MAPKAP K2/3, PRAK, MNK 1/2, MSK1/RLPK, RSK-B),transcription factors (ATF2/6, myocyte enhancer factor 2, nucleartranscription factor-β, CHOP/GADD153, Elk1 and SAP-1A1) and cytosolicproteins (stathmin), many of which are important physiologically.

Jiang, Y. et al. 1996 J Biol Chem 271:17920-17926 reportedcharacterization of p38β as a 372-amino acid protein closely related top38-α. Both p38α and p38β are activated by proinflammatory cytokines andenvironmental stress, p38β is preferentially activated by MAP kinasekinase-6 (MKK6) and preferentially phosphohorylate activatedtranscription factor 2 (ATF2). Kumar, S. et al. 1997 Biochem Biophys ResComm 235:533-538 and Stein, B. et al. 1997 J Biol Chem 272:19509-19517reported a second isoform of p38β, p-38β2, containing 364 amino acidswith 73% identity to p38α. It is believed that p38β is activated byproinflammatory cytokines and environmental stress, although the secondreported p38β isoform, p38β2, appears to be preferentially expressed inthe central nervous system (CNS), heart and skeletal muscle, compared tothe more ubiquitous tissue expression of p38α. Furthermore, it isbelieved that activated transcription factor-2 (ATF-2) is a bettersubstrate for p38β2 than for p38α.

The identification of p38γ was reported by Li, Z. et al. 1996 BiochemBiophys Res Comm 228:334-340 and of p38δ by Wang, X. et al. 1997 J BiolChem 272:23668-23674 and by Kumar, S. et al. 1997 Biochem Biophys ResComm 235:533-538. These two p38 isoforms (γ and δ) represent a uniquesubset of the MAPK family based on their tissue expression patterns,substrate utilization, response to direct and indirect stimuli, andsusceptibility to kinase inhibitors. Based upon primary sequenceconservation, p38α and β are closely related, but diverge from γ and δ,which are more closely related to each other.

Typically the p38 MAP kinase pathway is directly or indirectly activatedby cell surface receptors, such as receptor tyrosine kinases, chemokineor G protein-coupled receptors, which have been activated by a specificligand, e.g., cytokines, chemokines or lipopolysaccharide (LPS) bindingto a cognate receptor. Subsequently, a p38 MAP kinase is activated byphosphorylation on specific threonine and tyrosine residues. Afteractivation, p38 MAP kinase can phosphorylate other intracellularproteins, including protein kinases, and can be translocated to the cellnucleus, where it phosphorylates and activates transcription factorsleading to the expression of pro-inflammatory cytokines and otherproteins that contribute to the inflammatory response, cell adhesion,and proteolytic degradation. For example, in cells of myeloid lineage,such as macrophages and monocytes, both IL-1β and TNFα are transcribedin response to p38 activation. Subsequent translation and secretion ofthese and other cytokines initiates a local or systemic inflammatoryresponse in adjacent tissue and through infiltration of leukocytes.While this response is a normal part of physiological responses tocellular stress, acute or chronic cellular stress leads to the excess,unregulated, or excess and unregulated expression of pro-inflammatorycytokines. This, in turn, leads to tissue damage, often resulting inpain and debilitation.

In alveolar macrophages, inhibition of p38 kinases with p38 inhibitor,SB203580, reduces cytokine gene products. It is believed thatinflammatory cytokines (TNF-α, IFN-γ, IL-4, IL-5) and chemokines (IL-8,RANTES, eotaxin) are capable of regulating or supporting chronic airwayinflammation. The production and action of many of the potentialmediators of airway inflammation appear to be dependent upon thestress-activated MAP kinase system (SAPK) or p38 kinase cascade(Underwood et al. 2001 Frog Respir Res 31:342-345). Activation of thep38 kinase pathway by numerous environmental stimuli results in theelaboration of recognized inflammatory mediators whose production isconsidered to be translationally regulated. In addition, a variety ofinflammatory mediators activate p38 MAPK which may then activatedownstream targets of the MAPK system including other kinases ortranscription factors, thus creating the potential for an amplifiedinflammatory process in the lung.

Downstream Substrates of P38 Group of Map Kinases

Protein kinase substrates of p38α or p38β: MAP kinase-activated proteinkinase 2 (MAPKAPK2 or M2), MAP kinase interaction protein kinase (MNK1),p38 regulated/activated kinase (PRAK), mitogen- and stress-activatedkinase (MSK: RSK-B or RLPK).

Transcription factors activated by p38: activating transcription factor(ATF)-1, 2 and 6, SRF accessory protein 1 (Sap 1), CHOP (growth arrestand DNA damage inducible gene 153, or GADD153), p53, C/EBPβ, myocyteenhance factor 2C (MEF2C), MEF2A, MITF1, DDIT3, ELK1, NFAT, and highmobility group-box protein (HBP1).

Other types of substrates for p38: cPLA2, Na⁺/H⁺ exchanger isoform-1,tau, keratin 8, and stathmin.

Genes regulated by the p38 pathway: c-jun, c-fos, junB, IL-1, TNF, IL-6,IL-8, MCP-1, VCAM-1, iNOS, PPARγ, cyclooxygenase (COX)-2, collagenase-1(MMP-1), Collagenase-3 (MMP-13), HIV-LTR, Fgl-2, brain natriureticpeptide (BNP), CD23, CCK, phosphoenolpyruvate carboxy-kinase-cytosolic,cyclin D1, LDL receptor (Ono et al. 2000 Cellular Signalling 12:1-13).

Biological Consequences of p38 Activation p38 and Inflammation

Acute and chronic inflammation are believed to be central to thepathogenesis of many diseases such as rheumatoid arthritis, asthma,chronic obstructive pulmonary disease (COPD) and acute respiratorydistress syndrome (ARDS). The activation of the p38 pathway may play ancentral role in: (1) production of proflammatory cytokines such asIL-1β, TNF-α and IL-6; (2) induction of enzymes such as COX-2, whichcontrols connective tissue remodeling in pathological condition; (3)expression of an intracellular enzyme such as iNOS, which regulatesoxidation; (4) induction of adherent proteins such as VCAM-1 and manyother inflammatory related molecules. In addition to these, the p38pathway may play a regulatory role in the proliferation anddifferentiation of cells of the immune system. p38 may participate inGM-CSF, CSF, EPO, and CD40-induced cell proliferation and/ordifferentiation.

The role of the p38 pathway in inflammatory-related diseases was studiedin several animal models. Inhibition of p38 by SB203580 reducedmortality in a murine model of endotoxin-induced shock and inhibited thedevelopment of mouse collagen-induced arthritis and rat adjuvantarthritis. A recent study showed that SB220025, which is a more potentp38 inhibitor, caused a significant dose-dependent decrease in vasculardensity of the granuloma. These results indicate that p38 or thecomponents of the p38 pathway can be a therapeutic target forinflammatory disease.

p38 and Apoptosis

It appears that concomitant activation of p38 and apoptosis is inducedby a variety of agents such as NGF withdrawal and Fas ligation. Cysteineproteases (caspases) are central to the apoptotic pathway and areexpressed as inactive zymogens. Caspase inhibitors may then block p38activation through Fas cross-linking. However, overexpression ofdominant active MKK6b can also induce caspase activity and cell death.The role of p38 in apoptosis is cell type- and stimulus-dependent. Whilep38 signaling has been shown to promote cell death in some cell lines,in different cell lines p38 has been shown to enhance survival, cellgrowth, and differentiation.

p38 in the Cell Cycle

Overexpression of p38α in yeast leads to significant slowing ofproliferation, indicating involvement of p38α in cell growth. A slowerproliferation of cultured mammalian cells was observed when the cellswere treated with p38α/β inhibitor, SB203580.

p38 and Cardiomyocyte Hypertrophy

Activation and function of p38 in cardiomyocyte hypertrophy has beenstudied. During progression of hypertrophy, both p38α and p38β levelswere increased and constitutively active MKK3 and MKK6-elicitedhypertrophic responses enhanced by sarcomeric organization and elevatedatrial natriuretic factor expression. Also, reduced signaling of p38 inthe heart promotes myocyte differentiation via a mechanism involvingcalcineurin-NFAT signaling.

p38 and Development

Despite the non-viability of p38 knockout mice, evidence existsregarding the differential role of p38 in development. p38 has beenlinked to placental angiogenesis but not cardiovascular development inseveral studies. Furthermore, p38 has also been linked to erythropoietinexpression suggesting a role in erythropoiesis. PRAK has recently beenimplicated in cell development in murine implantation. PRAK mRNA, aswell as p38 isoforms, were found to be expressed throughout blastocystdevelopment

p38 and Cell Differentiation

p38α and/or p38β were found to play an important role in celldifferentiation for several different cell types. The differentiation of3T3-L1 cells into adipocytes and the differentiation of PC12 cells intoneurons both require p38α and/or β. The p38 pathway was found to benecessary and sufficient for SKT6 differentiation into hemoglobinizedcells as well as C2C112 differentiation in myotubules.

p38 in Senescence and Tumor Suppression

p38 has a role in tumorigenesis and senescence. There have been reportsthat activation of MKK6 and MKK3 led to a senescent phenotype dependentupon p38 MAPK activity. Also, p38 MAPK activity was shown responsiblefor senescence in response to telomere shortening, H₂O₂ exposure, andchronic RAS oncogene signaling. A common feature of tumor cells is aloss of senescence and p38 is linked to tumorigenesis in certain cells.It has been reported that p38 activation is reduced in tumors and thatloss of components of the p38 pathway such as MKK3 and MKK6 resulted inincreased proliferation and likelihood of tumorigenic conversionregardless of the cell line or the tumor induction agent used in thesestudies.

p38 MAP Kinase Inhibitors

A “p38 MAPK inhibitor” is a compound that inhibits the activity of p38.The inhibitory effects of a compound on the activity of p38 may bemeasured by various methods well-known to a skilled artisan. Forexample, the inhibitory effects may be measured by measuring the levelof inhibition of lipopolysaccharide (LPS)-stimulated cytokine production(Lee et al. 1988 Int J Immunopharmacol 10:835-843; Lee et al. 1993 AnnNY Acad Sci 696:149-170; Lee et al. 1994 Nature 372:739-746; Lee et al.1999 Pharmacol Ther 82:389-397).

Efforts to develop p38 MAPK inhibitors have focused on increasingpotency. SB203580 and other 2,4,5-triaryl imidazoles were found to bepotent p38 kinase inhibitors with EC₅₀ values in nanomolar range. Forexample, for SB203580 the EC₅₀ was found to be 48 nM. Thepyridinylimidazoles SKF 86002 (P1) and SB203582 (P2) shown below havebeen used as the template for the majority of p38 inhibitors. Recentpublications (Lee et al. 2000 Immunopharmacology 47:185-201) havedisclosed the p38 inhibitors (P3-P6) shown below. Notable among theseinhibitors is the relatively high potency and selectivity described forcompound P4 (p38 EC₅₀=0.19 nM) and the inhibition of inflammation drivenangiogenesis by SB 220025 (P6).

Two p38 inhibitors reported to be in clinical development are HEP689(P7) and VX-745 (P8). VX-745 is reportedly in Phase II trials forrheumatoid arthritis. Potent topical anti-inflammatory activity has beendisclosed for HRP689, which has reportedly entered clinical developmentto explore its potential as a topical agent for the treatment ofpsoriasis and other skin disorders.

Further discussion of various p38 inhibitors can be found in Boehm etal. 2000 Exp Opin Ther Pat 10:25-37; and Salituro et al. 1999 Curr MedChem 6:807-823.

Preferred p38 inhibitors described herein are pirfenidone derivativesand analogs that exhibit relatively low potency of p38 inhibition while,surprisingly, still having a relatively high therapeutic effect (e.g.,for modulating an SAPK system) as a result of such inhibition.Preferably, the p38 inhibitors of the embodiments exhibit EC₅₀ in therange of about 1 μM and about 1000 μM, preferably about 50 μM to about650 μM for the inhibition of the p38 MAPK.

Pirfenidone Derivatives and Analogs

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) itself is a knowncompound and its pharmacological effects are disclosed, for example, inJapanese Patent Application KOKAI (Laid-Open) Nos. 87677/1974 and1284338/1976. U.S. Pat. No. 3,839,346, issued Oct. 1, 1974; U.S. Pat.No. 3,974,281, issued Aug. 10, 1976; U.S. Pat. No. 4,042,699, issuedAug. 16, 1977; and U.S. Pat. No. 4,052,509, issued Oct. 4, 1977, all ofwhich are hereby incorporated by reference in their entireties, describemethods of manufacture of 5-methyl-1-phenyl-2-(1H)-pyridone and its useas an anti-inflammatory agent.

Pirfenidone and derivatives thereof are useful compounds for modulatinga stress activated protein kinase (SAPK) system.

The term “alkyl” used herein refers to a monovalent straight or branchedchain radical of from one to ten carbon atoms, including, but notlimited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

The term “alkenyl” used herein refers to a monovalent straight orbranched chain radical of from two to ten carbon atoms containing acarbon double bond including, but not limited to, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like.

The term “halogen” used herein refers to fluorine, chlorine, bromine, oriodine.

The term “haloalkyl” used herein refers to one or more halogen groupsappended to an alkyl radical.

The term “nitroalkyl” used herein refers to one or more nitro groupsappended to an alkyl radical.

The term “thioalkyl” used herein refers to one or more thio groupsappended to an alkyl radical.

The term “hydroxyalkyl” used herein refers to one or more hydroxy groupsappended to an alkyl radical.

The term “alkoxy” used herein refers to straight or branched chain alkylradical covalently bonded to the parent molecule through an —O— linkage.Examples of alkoxy groups include, but are limited to, methoxy, ethoxy,propoxy, isopropoxy, butoxy, n-butoxy, sec-butoxy, t-butoxy and thelike.

The term “alkoxyalkyl” used herein refers to one or more alkoxy groupsappended to an alkyl radical.

The term “carboxy” used herein refers to —COOH optionally appended to analkyl group. Examples of carboxy groups include, but are not limited to,—COOH, —CH₂COOH, —CH₂CH₂COOH, —CH(COOH)(CH₃), and the like.

The term “alkoxycarbonyl” refers to —(CO)—O-alkyl. Examples ofalkoxycarbonyl groups include, but are limited to, methoxycarbonylgroup, ethoxycarbonyl group, propoxycarbonyl group, and the like.

Carbohydrates are polyhydroxy aldehydes or ketones, or substances thatyield such compounds upon hydrolysis. Carbohydrates comprise theelements carbon (C), hydrogen (H) and oxygen (O) with a ratio ofhydrogen twice that of carbon and oxygen.

In their basic form, carbohydrates are simple sugars or monosaccharides.These simple sugars can combine with each other to form more complexcarbohydrates. The combination of two simple sugars is a disaccharide.Carbohydrates consisting of two to ten simple sugars are calledoligosaccharides, and those with a larger number are calledpolysaccharides.

The term “uronide” refers to a monosaccharide having a carboxyl group(—COOH) on the carbon that is not part of the ring. The uronide nameretains the root of the monosaccharide, but the -ose sugar suffix ischanged to -uronide. For example, the structure of glucuronidecorresponds to glucose.

As used herein, a radical indicates species with a single, unpairedelectron such that the species containing the radical can be covalentlybonded to another species. Hence, in this context, a radical is notnecessarily a free radical. Rather, a radical indicates a specificportion of a larger molecule. The term “radical” can be usedinterchangeably with the term “group.”

As used herein, a substituted group is derived from the unsubstitutedparent structure in which there has been an exchange of one or morehydrogen atoms for another atom or group. When substituted, thesubstituent group(s) is (are) one or more group(s) individually andindependently selected from C₁-C₁₀ alkyl, C₁-C₁₀ cycloalkyl, aryl, fusedaryl, heterocyclyl, heteroaryl, hydroxy, C₁-C₁₀ alkoxy, aryloxy,mercapto, C₁-C₁₀ alkylthio, arylthio, cyano, halogen, carbonyl,thiocarbonyl, C₁-C₁₀ alkoxycarbonyl, nitro, silyl,trihalomethanesulfonyl, trifluoromethyl, and amino, including mono- anddi-substituted amino groups, and the protected derivatives thereof. Theprotecting groups that can form the protective derivatives of the abovesubstituents are known to those of skill in the art and can be found inreferences such as Greene and Wuts Protective Groups in OrganicSynthesis; John Wiley and Sons: New York, 1999. Wherever a substituentis described as “optionally substituted” that substituent can besubstituted with the above substituents.

The term “purified” refers to a compound which has been separated fromother compounds such that it comprises at least 95% of the measuredsubstance when assayed.

Asymmetric carbon atoms may be present in the compounds describedherein. All such isomers, including diastereomers and enantiomers, aswell as the mixtures thereof are intended to be included in the scope ofthe recited compound. In certain cases, compounds can exist intautomeric forms. All tautomeric forms are intended to be included inthe scope of the recited compound. Likewise, when compounds contain analkenyl or alkenylene group, there exists the possibility of cis- andtrans-isomeric forms of the compounds. Both cis- and trans-isomers, aswell as the mixtures of cis- and trans-isomers, are contemplated. Thus,reference herein to a compound includes all of the aforementionedisomeric forms unless the context clearly dictates otherwise.

Various forms are included in the embodiments, including polymorphs,solvates, hydrates, conformers, salts, and prodrug derivatives. Apolymorph is a composition having the same chemical formula, but adifferent structure. A solvate is a composition formed by solvation (thecombination of solvent molecules with molecules or ions of the solute).A hydrate is a compound formed by an incorporation of water. A conformeris a structure that is a conformational isomer. Conformational isomerismis the phenomenon of molecules with the same structural formula butdifferent conformations (conformers) of atoms about a rotating bond.Salts of compounds can be prepared by methods known to those skilled inthe art. For example, salts of compounds can be prepared by reacting theappropriate base or acid with a stoichiometric equivalent of thecompound. A prodrug is a compound that undergoes biotransformation(chemical conversion) before exhibiting its pharmacological effects. Forexample, a prodrug can thus be viewed as a drug containing specializedprotective groups used in a transient manner to alter or to eliminateundesirable properties in the parent molecule. Thus, reference herein toa compound includes all of the aforementioned forms unless the contextclearly dictates otherwise.

The compounds described below are useful in the methods describedherein. In an embodiment, a compound as described below exhibits an EC₅₀in the range of about 1 μM to about 1000 μM for inhibition of p38 MAPK.

An embodiment provides a family of compounds represented by thefollowing genus (Genus Ia):

wherein

R₁, R₂, R₃, and R₄ are independently selected from the group consistingof H, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀haloalkyl, C₁-C₁₀ nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀ hydroxyalkyl,C₁-C₁₀ alkoxy, phenyl, substituted phenyl, halogen, hydroxyl, C₁-C₁₀alkoxyalkyl, C₁-C₁₀ carboxy, C₁-C₁₀ alkoxycarbonyl, CO-uronide,CO-monosaccharide, CO-oligosaccharide, and CO-polysaccharide; and

X₁, X₂, X₃, X₄, and X₅ are independently selected from the groupconsisting of H, halogen, alkoxy, and hydroxy.

Another embodiment provides a family of compounds represented by thefollowing genus (Genus Ib):

wherein

X₃ is selected from the group consisting of H, halogen, C₁-C₁₀ alkoxy,and OH;

R₂ is selected from the group consisting of H, halogen, C₁-C₁₀ alkyl,substituted C₁-C₁₀ alkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ alkoxyalkyl,C₁-C₁₀ carboxy, C₁-C₁₀ alkoxycarbonyl, CO-uronide, CO-monosaccharide,CO-oligosaccharide, and CO-polysaccharide; and

R₄ is selected from the group consisting of H, halogen, and OH.

Another embodiment provides a family of compounds represented by thefollowing genus (Genus Ic):

wherein

X₃ is selected from the group consisting of H, F, OH, and OCH₃;

R₂ is selected from the group consisting of H, CF₃, CHF₂, CH₂F, CH₂OH,COOH, CO-Glucoronide, Br, CH₃, and CH₂OCH₃; and

R₄ is selected from the group consisting of H and OH;

with the proviso that when R₄ and X₃ are H, R₂ is not CH₃

Another embodiment provides a family of compounds represented by thefollowing subgenus (Subgenus II):

wherein

is selected from the group consisting of H, OH, and OCH₃;

R₂ is selected from the group consisting of H, CH₂OH, COOH,CO-Glucoronide, Br, CH₃, and CH₂OCH₃; and

R₄ is selected from the group consisting of H and OH, with the provisothat when X₃ is OH then R₂ is not CH₃.

Another embodiment provides a family of compounds represented by thefollowing subgenus (Subgenus III):

wherein

X₃ is selected from the group consisting of H, F, and OH; and

R₂ is selected from the group consisting of H, Br, CH₂F, CHF₂, and CF₃.

Another embodiment provides a family of compounds represented by thefollowing subgenus (Subgenus IV):

wherein X₃ is selected from the group consisting of H, halogen, C₁-C₁₀alkoxy, OH, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl,C₁-C₁₀ haloalkyl, C₁-C₁₀ nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀hydroxyalkyl, phenyl, substituted phenyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀carboxy, C₁-C₁₀ alkoxycarbonyl, CO-uronide, CO-monosaccharide,CO-oligosaccharide, and CO-polysaccharide.

Another embodiment provides a family of compounds represented by thefollowing subgenus (Subgenus V):

wherein X₃ is selected from the group consisting of H, halogen, C₁-C₁₀alkoxy, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀haloalkyl, C₁-C₁₀ nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀ hydroxyalkyl,phenyl, substituted phenyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ carboxy, C₁-C₁₀alkoxycarbonyl, CO-uronide, CO-monosaccharide, CO-oligosaccharide, andCO-polysaccharide.

Another embodiment provides a family of compounds represented by thefollowing genus (Genus VI):

wherein

Ar is pyridinyl or phenyl;

Z is O or S;

X₃ is H, F, Cl, OH, CH₃, or OCH₃;

R₂ is methyl, C(═O)H, C(═O)CH₃, C(═O)OCH₃, C(═O)O-glucosyl,fluoromethyl, difluoromethyl, trifluoromethyl, bromo, methylmethoxyl,methylhydroxyl, or phenyl; and

R₄ is H or hydroxyl;

with the proviso that when R₂ is trifluoromethyl, Z is O, R₄ is H and Aris phenyl, the phenyl is not solely substituted at the 4′ position by H,F, or OH.

The Genus VI includes the families of compounds represented by theSubgenus VIa and the Subgenus VIb:

wherein Z, X₃, R₂ and R₄ are defined as in Genus VI. It will berecognized that the phenyl ring in the structure represented by SubgenusVIa is substituted by X₃ at the 4′ position.

Another embodiment provides a family of compounds represented by thefollowing genus (Genus VII):

wherein

X₃ is selected from the group consisting of H, halogen, C₁-C₁₀ alkoxy,and OH;

Y₁, Y₂, Y₃, and Y₄ are independently selected from the group consistingof H, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀haloalkyl, C₁-C₁₀ nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀ hydroxyalkyl,C₁-C₁₀ alkoxy, phenyl, substituted phenyl, halogen, hydroxyl, C₁-C₁₀alkoxyalkyl, C₁-C₁₀ carboxy, C₁-C₁₀ alkoxycarbonyl; and

R₄ is selected from the group consisting of H, halogen, and OH.

It will be recognized that a particular compound described herein may bea member of more than one of the various genera described above. Thecompounds described herein are useful for modulating a stress activatedprotein kinase (SAPK) system. Exemplary compounds of Genera Ia-c,Subgenera II-V and Genera VI and VII that are useful for modulating astress activated protein kinase (SAPK) system are set forth in Table 1below. Compounds 1-6 are examples of compounds of Subgenus II. Compounds7-12 are examples of compounds of Subgenus III. Compound 13 ispirfenidone, an example of a compound of Subgenus II. Compounds 14-32are examples of compounds of Genus VI. Compound 33 is an example ofGenus VII.

TABLE 1 Compound Number Compound 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

Another embodiment is directed to purified compounds represented byGenera Ia-c, Subgenera II-V and/or Genera VI and VII. The degree ofpurity may be expressed as a percentage as described above. In preferredembodiments, purified compounds represented by Genera Ia-c, SubgeneraII-V and/or Genera VI and VII have a purity of about 96% or greater,more preferably about 98% or greater, by weight based on total weight ofthe composition that comprises the purified compound. For example, anembodiment provides purified Compound 3 (Table 1).

Compounds of Genera Ia-c, Subgenera II-V and/or Genera VI and VII can besynthesized by using various reactions. Examples of syntheses includethe following, designated Synthetic Schemes 1, 2, and 3.

Ullmann reaction: Chem. Pharm. Bull. 45(4) 719-721. TargetN-aryl-pyridine-2-ones were obtained via arylation of2-hydroxypyridines. The Ullmann reaction is useful in the preparation ofdisclosed compounds, except the 5-bromo analogs and compound 33 whichare afforded, for example, by synthetic scheme 3.

A mixture of 2-hydroxypyridine (1 mmol), aryl iodide or bromide (2mmol), CuI (0.1-0.5 mmol) and anhydrous potassium carbonate (1 mmol) inDMF (3 ml) was stirred overnight at 135° C. under argon atmosphere. Deepcolored reaction mixture was taken into ethyl acetate and 10% ammoniumhydroxide. Organic layer was washed with brine and dried over magnesiumsulfate. Column chromatography furnished target compounds as off-whitesolids in 25-60% yield.

Target N-aryl-2-pyridones may be obtained via arylation of2-hydroxypyridines with alkylboronic acids (Tetrahedron Lett., 42 (2001)3415-3418). The alkylboronic acid route is useful in the preparation ofdisclosed compounds. A mixture of 2-hydroxypyridine (5 mmol),arylboronic acid (10 mmol), cupper (1 l) acetate (0.5-1 mmol), pyridine(10 mmol) and molecular sieves 4A (0.5-1 g) in dichloromethane (25 ml)was stirred for 24-48 hours at room temperature opened to the air.Reaction mixture was washed with saturated sodium bicarbonate with EDTAand organic phase was dried over sodium sulfate. TargetN-aryl-2-pyridones were isolated by column chromatography as whitesolids in 85-100% yield.

As pirfenidone derivatives, compounds of Genera Ia-c, Subgenera II-Vand/or Genera VI and VII may also be synthesized by any conventionalreactions known in the art based on the known synthetic schemes forpirfenidone, such as disclosed in U.S. Pat. Nos. 3,839,346; 3,974,281;4,042,699; and 4,052,509, all of which are hereby expressly incorporatedby reference in their entirety.

Starting materials described herein are available commercially, areknown, or can be prepared by methods known in the art. Additionally,starting materials not described herein are available commercially, areknown, or can be prepared by methods known in the art.

Starting materials can have the appropriate substituents to ultimatelygive desired products with the corresponding substituents.Alternatively, substituents can be added at any point of synthesis toultimately give desired products with the corresponding substituents.

Synthetic Schemes 1-3 show methods that can be used to prepare compoundsof Genera Ia-c, Subgenera II-V and/or Genera VI and VII. One skilled inthe art will appreciate that a number of different synthetic reactionschemes can be used to synthesize the compounds of Genera Ia-c,Subgenera II-V and/or Genera VI and VII. Further, one skilled in the artwill understand that a number of different solvents, coupling agents,and reaction conditions can be used in the syntheses reactions to yieldcomparable results.

One skilled in the art will appreciate variations in the sequence and,further, will recognize variations in the appropriate reactionconditions from the analogous reactions shown or otherwise known whichmay be appropriately used in the processes above to make the compoundsof Genera Ia-c, Subgenera II-V and/or Genera VI and VII.

In the processes described herein for the preparation of the compoundsof compounds of Genera Ia-c, Subgenera II-V and/or Genera VI and VII,the use of protective groups is generally well recognized by one skilledin the art of organic chemistry, and accordingly the use of appropriateprotecting groups may in some cases be implied by the processes of theschemes herein, although such groups may not be expressly illustrated.Introduction and removal of such suitable protecting groups are wellknown in the art of organic chemistry; see for example, T. W. Greene,“Protective Groups in Organic Synthesis”, Wiley (New York), 1999. Theproducts of the reactions described herein may be isolated byconventional means such as extraction, distillation, chromatography, andthe like.

The salts, e.g., pharmaceutically acceptable salts, of the compounds ofGenera Ia-c, Subgenera II-V and/or Genera VI and VII may be prepared byreacting the appropriate base or acid with a stoichiometric equivalentof the compounds. Similarly, pharmaceutically acceptable derivatives(e.g., esters), metabolites, hydrates, solvates and prodrugs of thecompounds of Genera Ia-c, Subgenera II-V and/or Genera VI and VII may beprepared by methods generally known to those skilled in the art. Thus,another embodiment provides compounds that are prodrugs of an activecompound. In general, a prodrug is a compound which is metabolized invivo (e.g., by a metabolic transformation such as deamination,dealkylation, de-esterification, and the like) to provide an activecompound. A “pharmaceutically acceptable prodrug” means a compound whichis, within the scope of sound medical judgment, suitable forpharmaceutical use in a patient without undue toxicity, irritation,allergic response, and the like, and effective for the intended use,including a pharmaceutically acceptable ester as well as a zwitterionicform, where possible, of the compounds of the embodiments. Examples ofpharmaceutically-acceptable prodrug types are described in T. Higuchiand V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of theA.C.S. Symposium Series, and in Edward B. Roche, ed., BioreversibleCarriers in Drug Design, American Pharmaceutical Association andPergamon Press, 1987, both of which are hereby expressly incorporated byreference in their entirety.

The compounds and compositions described herein may also includemetabolites. As used herein, the term “metabolite” means a product ofmetabolism of a compound of the embodiments or a pharmaceuticallyacceptable salt, analog, or derivative thereof, that exhibits a similaractivity in vitro or in vivo to a compound of the embodiments. Thecompounds and compositions described herein may also include hydratesand solvates. As used herein, the term “solvate” refers to a complexformed by a solute (herein, a compound of Genera Ia-c, Subgenera II-Vand/or Genera VI and VII) and a solvent. Such solvents for the purposeof the embodiments preferably should not interfere with the biologicalactivity of the solute. Solvents may be, by way of example, water,ethanol, or acetic acid. In view of the foregoing, reference herein to aparticular compound or genus of compounds will be understood to includethe various forms described above, including pharmaceutically acceptablesalts, esters, prodrugs, metabolites and solvates thereof unless statedotherwise.

Methods of Inhibiting p38 MAP Kinase

In an embodiment, methods are provided for modulating a SAPK system, invitro or in vivo. The methods include contacting a SAPK-modulatingconcentration of a compound with at least one p38 MAPK (e.g., bycontacting the compound with a cell or tissue containing at least onep38 MAPK), where the compound has a relatively low potency forinhibition of the at least one p38 MAPK, corresponding to a relativelyhigh inhibitory concentration for inhibition of the at least one p38MAPK by the compound.

“Contacting a cell” refers to a condition in which a compound or othercomposition of matter is in direct contact with a cell or tissue, or isclose enough to induce a desired biological effect in a cell or tissue.For example, contacting a cell or tissue containing p38 MAPK with acompound may be conducted in any manner that permits an interactionbetween p38 MAPK and the compound, resulting in the desired biologicaleffect in a cell. Contacting a cell or tissue may be accomplished, forexample, by intermixing or administration of a compound (such as acompound of Genera Ia-c, Subgenera II-V and/or Genera VI and VII and/ora salt, ester, prodrug and/or intermediate thereof, and/or apharmaceutical composition comprising one or more of the foregoing).

Alternatively, contacting a cell or tissue may be accomplished byintroducing a compound in a manner such that the compound will betargeted, directly or indirectly, to a cell or tissue containing p38MAPK. Contacting a cell or tissue may be accomplished under conditionssuch that a compound binds to at least one p38 MAPK. Such conditions mayinclude proximity of the compound and p38-containing cell or tissue, pH,temperature, or any condition that affects the binding of a compound top38 MAPK.

In certain embodiments, the cell is contacted with the compound invitro; in other embodiments, the cell is contacted with the compound invivo.

When the cell is contacted in vivo, the effective concentration (EC) ofa compound is a concentration that results in a reduction of a specifiedendpoint by a target percentage (e.g., 50%, 40%, 30%, 20%, 10%) relativeto the maximal observable reduction of the specified endpoint by thatcompound. Such an endpoint may be a physiological response, for example,reduction in blood or other bodily fluid concentration of TNFα. Forexample, EC₅₀, EC₄₀, EC₃₀, EC₂₀ and EC₁₀ are determined asconcentrations that result in reductions in the serum TNFα concentrationby 50%, 40%, 30%, 20% and 10%, respectively, relative to the maximalobservable reduction on a dose-response curve.

When the cell is contacted in vitro, except in a cell-based assay, theeffective concentration (EC) is a concentration that results in areduction in the activity of the specified target by a given percentage(e.g., 50%, 40%, 30%, 20%, 10%). For example, EC₅₀, EC₄₀, EC₃₀, EC₂₀ andEC₁₀ are determined as concentrations that result in reductions in theactivity of the specified target by 50%, 40%, 30%, 20% and 10%,respectively, on a dose-response curve. When complete inhibition of aspecified target is not obtained, the effective concentration (EC) of acompound is a concentration that results in a reduction of a targetactivity by a given percentage (e.g., 50%, 40%, 30%, 20%, 10%) relativeto the maximal reduction of the target activity by that compound.

When the cell is contacted in vitro, in a cell-based assay, theeffective concentration (EC) of a compound is a concentration thatresults in a reduction of a specified endpoint by a target percentage(e.g., 50%, 40%, 30%, 20%, 10%) relative to the maximal observablereduction of the specified endpoint by that compound. Such an endpointmay be a cellular response, for example, reduction in the secretion ofTNFα as determined by the TNFα concentration in cell medium. Forexample, EC₅₀, EC₄₀, EC₃₀, EC₂₀ and EC₁₀ are determined asconcentrations that result in reductions in the TNFα concentration by50%, 40%, 30%, 20% and 10%, respectively, relative to the maximalobservable reduction on a dose-response curve.

The EC₅₀ of the SAPK system-modulating compound is preferably in therange of about 1 μM to about 1000 μM, more preferably about 50 μM toabout 650 μM for inhibition of at least one p38 MAPK. Thus, for example,modulation of the SAPK system may involve contacting a compound (e.g., acompound of Genera Ia-c, Subgenera II-V and/or Genera VI and VII) withat least one p38 MAPK at a concentration that is less than an EC₄₀,preferably less than EC₃₀, more preferably less than EC₂₀, evenpreferably less than EC₁₀ for inhibition of the at least one p38 MAPK bythe compound as determined on a dose-response curve in vivo.

In certain embodiments, the compound is provided in the form of apharmaceutical composition, together with a pharmaceutically acceptablecarrier.

Screening a Library of Compounds for Low-Potency p38 Inhibitors

In another aspect, a method is provided for identifying apharmaceutically active compound, e.g., for determining whether acompound is potentially useful as a therapeutic agent, e.g., for theprevention or treatment of an inflammatory condition (such as p38- orcytokine-associated condition). The method includes assaying a pluralityof compounds for inhibition of at least one p38 MAPK and selecting acompound which exhibits a relatively low potency for inhibiting p38MAPK. Preferably, an EC₅₀ of such a low-potency p38 inhibitor compoundis in the range of about 1 μM to about 1000 μM, preferably about 50 μMto about 650 μM for inhibition of the at least one p38 MAPK. Theplurality of compounds to be assayed is preferably selected from alibrary of potential compounds. The assaying of the plurality ofcompounds from the library may be conducted in various ways. Forexample, in some embodiments, the methods further comprise contacting atleast one p38 MAPK with the plurality of compounds, and determiningwhether the compounds inhibit the activity of cytokines. A p38 MAPK ispreferably selected from the group consisting of p38α, p38β, p38γ, andp38δ. In preferred embodiments, the contacting step takes place invitro; in certain preferred embodiments, the contacting step comprisescontacting a cell comprising p38 MAPK with the compound.

In yet another embodiment, methods are provided for inhibiting theactivity of p38 MAPK in a cell, in vitro or in vivo. In general, suchmethods include contacting a cell containing at least one p38 MAPK withan effective p38-inhibiting amount of a compound (e.g., a compound ofGenera Ia-c, Subgenera II-V and/or Genera VI and VII), under conditionssuch that p38 activity in the cell is inhibited. Examples of suchmethods are provided in the EXAMPLES section below. The compoundpreferably exhibits an EC₅₀ in the range of about 1 μM to about 1000 μM,preferably about 50 μM to about 650 μM for inhibition of the at leastone p38 MAPK. The contacting of at least one p38 MAPK with the compoundis preferably conducted at a SAPK system-modulating concentration thatis less than EC₃₀, preferably less than EC₂₀, more preferably less thanEC₁₀ for inhibition of the at least one p38 MAPK by the compound.

In vivo methods include for example, introducing into a group of animalsorally or by injection a compound of interest (e.g., a compound ofGenera Ia-c, Subgenera II-V and/or Genera VI and VII) in variousconcentrations. Following the introduction of the compound,lipopolysaccharide is administered intravenously. Serum TNFα levels aremeasured and compared to that from control animals. The preferredcompounds inhibit the release of TNFα, thus reducing TNFα levels in theblood samples of the tested animals. The compound preferably exhibits anEC₅₀ in the range of about 1 μM to about 1000 μM, preferably about 50 μMto about 650 μM for inhibition of the release of TNFα.

The method of identifying a pharmaceutically active compound may furtherinclude determining a mammalian toxicity of the selected compound. Suchmethods are generally known to those skilled in the art. The method ofidentifying a pharmaceutically active compound may also includeadministering the selected compound to a test subject, either inconjunction with the deteintination of mammalian toxicity or for otherreasons. In an embodiment, the test subject test subject has or is atrisk for having an inflammatory condition. Preferably the test subjectis a mammal, and may be a human.

Methods of Treatment and/or Prevention

Another embodiment provides methods for treating or preventing diseasestates, e.g., inflammatory condition(s) and/or fibrotic condition(s).The methods include identifying a subject at risk for or having at leastone condition selected from an inflammatory condition and a fibroticcondition and administering a compound to the subject in an effectiveamount to treat or prevent the inflammatory condition and/or fibroticcondition. In preferred embodiments, the compound exhibits an EC₅₀ inthe range of about 1 μM to about 1000 μM, preferably about 50 μM toabout 650 μM for inhibition of at least one p38 MAPK. In preferredembodiments, the effective amount produces a blood or serum or anotherbodily fluid concentration that is less than an EC₃₀ or, preferably, anEC₂₀ or, more preferably, an EC₁₀ for inhibition of p38 MAPK by thecompound. In preferred embodiments, the compound exhibits an EC₅₀ in therange of about 1 μM to about 1000 μM, preferably about 50 μM to about650 μM for inhibition of the TNFα secretion. In other preferredembodiments, the effective amount produces a blood or serum or anotherbodily fluid concentration that is less than an EC₃₀ or, preferably, anEC₂₀ or, more preferably, an EC₁₅ or, more preferably, an EC₁₀ forinhibition of LPS-stimulated TNFα release in a bodily fluid by thecompound. The effective amount is preferably about 70% or less, morepreferably less than about 50%, of an amount that causes an undesirableside effect in the subject, such as, but not limited to, drowsiness,nausea, cold symptom, gastrointestinal upset, and photosensitivity rash.The compound used for the treatment or prevention is preferably acompound of Genera Ia-c, Subgenera II-V and/or Genera VI and VII.

Methods for identifying a subject at risk for or having an inflammatorycondition are known to those skilled in the art. Examples ofinflammatory conditions that may be treated or prevented by the methodsdescribed herein include p-38 associated conditions, e.g., conditionsassociated with altered cytokine activity, conditions associated withmodulation of an SAPK system, autoimmune diseases, and diseasesassociated with acute and chronic inflammation. The cytokine (orcytokines) is (are) preferably selected from the group consisting of,but not limited to, IL-1β, IL-6, IL-8, and TNFα. In an embodiment, thecompound used to treat or prevent the inflammatory condition is compoundthat inhibits a kinase in the SAPK signaling pathway. Examples ofpreferred compounds include compound of Genera Ia-c, Subgenera II-Vand/or Genera VI and VII.

The term “p38-associated condition” means a disease or other deleteriouscondition in which the p38 MAP kinase signaling pathway is implicated,whether directly or indirectly. Examples of p38-associated conditionsinclude conditions caused by IL-1β, TNFα, IL-6 or IL-8 dysregulation oroverexpression resulting from sustained, prolonged, enhanced or elevatedlevels of p38 activity. Such conditions include, without limitation,inflammatory diseases, autoimmune diseases, fibrotic diseases,destructive bone disorders, proliferative disorders, infectiousdiseases, neurodegenerative diseases, allergies, reperfusion ischemia instroke, heart attacks, angiogenic disorders, organ hypoxia, vascularhyperplasia, cardiac hypertrophy, thrombin-induced platelet aggregation,and conditions associated with the prostaglandin or cyclooxygenasepathways, e.g., conditions involving prostaglandin endoperoxidesynthase. A p38-associated condition can include any conditionassociated with or mediated by an isoform of p38.

A “fibrotic condition,” “fibroproliferative condition,” “fibroticdisease,” “fibroproliferative disease,” “fibrotic disorder,” and“fibroproliferative disorder” are used interchangeably to refer to acondition, disease or disorder that is characterized by dysregulatedproliferation or activity of fibroblasts and/or pathologic or excessiveaccumulation of collagenous tissue. Typically, any such disease,disorder or condition is amenable to treatment by administration of acompound having anti-fibrotic activity. Fibrotic disorders include, butare not limited to, pulmonary fibrosis, including idiopathic pulmonaryfibrosis (IPF) and pulmonary fibrosis from a known etiology, liverfibrosis, and renal fibrosis. Other exemplary fibrotic conditionsinclude musculoskeletal fibrosis, cardiac fibrosis, post-surgicaladhesions, scleroderma, glaucoma, and skin lesions such as keloids.

The term “modulating SAPK system” means increasing or decreasingactivity of the stress-activated protein kinase system activity, e.g.,by inhibiting p38 activity, whether in vitro or in vivo. In certainembodiments, the SAPK system is modulated when p38 activity in a cell isinhibited by about 50%, preferably by about 40%, more preferably byabout 30%, even more preferably by about 20%, or yet even morepreferably by about 10% compared to the p38 activity of an untreatedcontrol cell.

A condition associated with altered cytokine activity, as used herein,refers to a condition in which cytokine activity is altered compared toa non-diseased state. This includes, but is not limited to, conditionscaused by IL-1β, TNFα, IL-6 or IL-8 overproduction or dysregulationresulting in sustained, prolonged, enhanced or elevated levels ofcytokine activity, which may be associated with p38 activity. Suchconditions include, without limitation, inflammatory diseases,autoimmune diseases, fibrotic diseases, destructive bone disorders,proliferative disorders, infectious diseases, neurodegenerativediseases, allergies, reperfusion/ischemia in stroke, heart attacks,angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiachypertrophy, thrombin-induced platelet aggregation, and conditionsassociated with the cyclooxygenase and lipoxygenase signaling pathways,such as prostaglandin endoperoxide synthase. A cytokine-associatedcondition can include any condition associated with or mediated by IL-1(particularly IL-1β), TNFα, IL-6 or IL-8, or any other cytokine whichcan be regulated by p38. In preferred embodiments, the cytokineassociated condition is a condition associated with TNFα.

The methods described herein may also be used to treat autoimmunediseases and diseases associated with acute and chronic inflammation.These diseases include, but are not limited to: chronic obstructivepulmonary disease (COPD), bronchiolitis obliterans syndrome, chronicallograft fibrosis, inflammatory pulmonary fibrosis (IPF), rheumatoidarthritis; rheumatoid spondylitis; osteoarthritis; gout, other arthriticconditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis;toxic shock syndrome; myofacial pain syndrome (MPS); Shigellosis;asthma; adult respiratory distress syndrome; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerularnephritis; scleroderma; chronic thyroiditis; Grave's disease; Oiniond'sdisease; autoimmune gastritis; myasthenia gravis; autoimmune hemolyticanemia; autoimmune neutropenia; thrombocytopenia; pancreatic fibrosis;chronic active hepatitis including hepatic fibrosis; acute and chronicrenal disease; renal fibrosis, irritable bowel syndrome; pyresis;restenosis; cerebral malaria; stroke and ischemic injury; neural trauma;Alzheimer's disease; Huntington's disease; Parkinson's disease; acuteand chronic pain; allergies, including allergic rhinitis and allergicconjunctivitis; cardiac hypertrophy, chronic heart failure; acutecoronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lymedisease; Reiter's syndrome; acute synoviitis; muscle degeneration,bursitis; tendonitis; tenosynoviitis; herniated, ruptured, or prolapsedintervertebral disk syndrome; osteopetrosis; thrombosis; silicosis;pulmonary sarcosis; bone resorption diseases, such as osteoporosis ormultiple myeloma-related bone disorders; cancer, including but notlimited to metastatic breast carcinoma, colorectal carcinoma, malignantmelanoma, gastric cancer, and non-small cell lung cancer;graft-versus-host reaction; and auto-immune diseases, such as MultipleSclerosis, lupus and fibromyalgia; AIDS and other viral diseases such asHerpes Zoster, Herpes Simplex I or II, influenza virus, Severe AcuteRespiratory Syndrome (SARS) and cytomegalovirus; and diabetes mellitus.In addition, the methods of the embodiments can be used to treatproliferative disorders (including both benign and malignanthyperplasias), including acute myelogenous leukemia, chronic myelogenousleukemia, Kaposi's sarcoma, metastatic melanoma, multiple myeloma,breast cancer, including metastatic breast carcinoma; colorectalcarcinoma; malignant melanoma; gastric cancer; non-small cell lungcancer (NSCLC); bone metastases, and the like; pain disorders includingneuromuscular pain, headache, cancer pain, dental pain, and arthritispain; angiogenic disorders including solid tumor angiogenesis, ocularneovascularization, and infantile hemangioma; conditions associated withthe cyclooxygenase and lipoxygenase signaling pathways, includingconditions associated with prostaglandin endoperoxide synthase-2(including edema, fever, analgesia, and pain); organ hypoxia;thrombin-induced platelet aggregation. In addition, the methodsdescribed herein may be useful for the treatment of protozoal diseasesin animals, including mammals.

A subject may include one or more cells or tissues, or organisms. Apreferred subject is a mammal. A mammal may include any mammal. As anon-limiting example, preferred mammals include cattle, pigs, sheep,goats, horses, camels, buffalo, cats, dogs, rats, mice, and humans. Ahighly preferred subject mammal is a human. The compound(s) may beadministered to the subject via any drug delivery route known in theart. Specific exemplary administration routes include oral, ocular,rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular,intravenous (bolus and infusion), intracerebral, transdermal, andpulmonary.

The terms “therapeutically effective amount” and “prophylacticallyeffective amount”, as used herein, refer to an amount of a compoundsufficient to treat, ameliorate, or prevent the identified disease orcondition, or to exhibit a detectable therapeutic, prophylactic, orinhibitory effect. The effect can be detected by, for example, theassays disclosed in the following examples. The precise effective amountfor a subject will depend upon the subject's body weight, size, andhealth; the nature and extent of the condition; and the therapeutic orcombination of therapeutics selected for administration. Therapeuticallyand prophylactically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of the clinician. Preferably, the effective amount of thecompound of the embodiments produces a blood or serum or another bodilyfluid concentration that is less than an EC₁₀, EC₂₀ or EC₁₀ forinhibition of p38 MAP kinase.

For any compound, the therapeutically or prophylactically effectiveamount can be estimated initially either in cell culture assays, e.g.,of neoplastic cells, or in animal models, usually rats, mice, rabbits,dogs, or pigs. The animal model may also be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred.However, the pharmaceutical compositions that exhibit narrow therapeuticindices are also within the scope of the embodiments. The data obtainedfrom cell culture assays and animal studies may be used in formulating arange of dosage for human use. The dosage contained in such compositionsis preferably within a range of circulating concentrations that includean ED₅₀ with little or no toxicity. The dosage may vary within thisrange depending upon the dosage form employed, sensitivity of thepatient, and the route of administration.

More specifically, the maximum plasma concentrations (C_(max)) may rangefrom about 65 μM to about 115 μM, or about 75 μM to about 105 μM, orabout 85 μM to about 95 μM, or about 85 μM to about 90 μM depending uponthe route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and is generallyavailable to practitioners in the art. In general the dose will be inthe range of about 100 mg/day to about 10 g/day, or about 200 mg toabout 5 g/day, or about 400 mg to about 3 g/day, or about 500 mg toabout 2 g/day, in single, divided, or continuous doses for a patientweighing between about 40 to about 100 kg (which dose may be adjustedfor patients above or below this weight range, particularly childrenunder 40 kg). Generally the dose will be in the range of about 25 mg/kgto about 300 mg/kg of body weight per day.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeagent(s) or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered twice a day, once a day,once very two days, three times a week, twice a week, every 3 to 4 days,or every week depending on half-life and clearance rate of theparticular formulation. For example, in an embodiment, a pharmaceuticalcomposition contains an amount of a compound as described herein that isselected for administration to a patient on a schedule selected from:twice a day, once a day, once very two days, three times a week, twice aweek, and once a week.

It will be appreciated that treatment as described herein includespreventing a disease, ameliorating symptoms, slowing diseaseprogression, reversing damage, or curing a disease.

In one aspect, treating an inflammatory condition results in an increasein average survival time of a population of treated subjects incomparison to a population of untreated subjects. Preferably, theaverage survival time is increased by more than about 30 days; morepreferably, by more than about 60 days; more preferably, by more thanabout 90 days; and even more preferably by more than about 120 days. Anincrease in survival time of a population may be measured by anyreproducible means. In a preferred aspect, an increase in averagesurvival time of a population may be measured, for example, bycalculating for a population the average length of survival followinginitiation of treatment with an active compound. In an another preferredaspect, an increase in average survival time of a population may also bemeasured, for example, by calculating for a population the averagelength of survival following completion of a first round of treatmentwith an active compound.

In another aspect, treating an inflammatory condition results in adecrease in the mortality rate of a population of treated subjects incomparison to a population of subjects receiving carrier alone. Inanother aspect, treating an inflammatory condition results in a decreasein the mortality rate of a population of treated subjects in comparisonto an untreated population. In a further aspect, treating aninflammatory condition results a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the embodiments, or apharmaceutically acceptable salt, metabolite, analog or derivativethereof. Preferably, the mortality rate is decreased by more than about2%; more preferably, by more than about 5%; more preferably, by morethan about 10%; and most preferably, by more than about 25%. In apreferred aspect, a decrease in the mortality rate of a population oftreated subjects may be measured by any reproducible means. In anotherpreferred aspect, a decrease in the mortality rate of a population maybe measured, for example, by calculating for a population the averagenumber of disease-related deaths per unit time following initiation oftreatment with an active compound. In another preferred aspect, adecrease in the mortality rate of a population may also be measured, forexample, by calculating for a population the average number of diseaserelated deaths per unit time following completion of a first round oftreatment with an active compound.

In another aspect, treating an inflammatory condition results in adecrease in growth rate of a tumor. Preferably, after treatment, tumorgrowth rate is reduced by at least about 5% relative to/number prior totreatment; more preferably, tumor growth rate is reduced by at leastabout 10%; more preferably, reduced by at least about 20%; morepreferably, reduced by at least about 30%; more preferably, reduced byat least about 40%; more preferably, reduced by at least about 50%; evenmore preferably, reduced by at least 60%; and most preferably, reducedby at least about 75%. Tumor growth rate may be measured by anyreproducible means of measurement. In a preferred aspect, tumor growthrate is measured according to a change in tumor diameter per unit time.

In another aspect, treating an inflammatory condition results in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at leastabout 5%; more preferably, by at least about 10%; more preferably, by atleast about 20%; more preferably, by at least about 30%; morepreferably, by at least about 40%; more preferably, by at least about50%; even more preferably, by at least about 60%; and most preferably,by at least about 75%. The rate of cellular proliferation may bemeasured by any reproducible means of measurement. In a preferredaspect, the rate of cellular proliferation is measured, for example, bymeasuring the number of dividing cells in a tissue sample per unit time.

In another aspect, treating an inflammatory condition results in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at leastabout 5%; more preferably, by at least about 10%; more preferably, by atleast about 20%; more preferably, by at least about 30%; morepreferably, by at least about 40%; more preferably, by at least about50%; even more preferably, by at least about 60%; and most preferably,by at least about 75%. The proportion of proliferating cells may bemeasured by any reproducible means of measurement. In a preferredaspect, the proportion of proliferating cells is measured, for example,by quantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. In another preferred aspect, theproportion of proliferating cells is equivalent to the mitotic index.

In another aspect, treating an inflammatory condition results in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least about 10%; morepreferably, reduced by at least about 20%; more preferably, reduced byat least about 30%; more preferably, reduced by at least about 40%; morepreferably, reduced by at least about 50%; even more preferably, reducedby at least about 60%; and most preferably, reduced by at least about75%. Size of an area or zone of cellular proliferation may be measuredby any reproducible means of measurement. In a preferred aspect, size ofan area or zone of cellular proliferation may be measured as a diameteror width of an area or zone of cellular proliferation.

The methods described herein may include identifying a subject in needof treatment. In a preferred embodiment, the methods include identifyinga mammal in need of treatment. In a highly preferred embodiment, themethods include identifying a human in need of treatment. Identifying asubject in need of treatment may be accomplished by any means thatindicates a subject who may benefit from treatment. For example,identifying a subject in need of treatment may occur by clinicaldiagnosis, laboratory testing, or any other means known to one of skillin the art, including any combination of means for identification.

As described elsewhere herein, the compounds described herein may beformulated in pharmaceutical compositions, if desired, and can beadministered by any route that permits treatment of the disease orcondition. A preferred route of administration is oral administration.Administration may take the form of single dose administration, or thecompound of the embodiments can be administered over a period of time,either in divided doses or in a continuous-release formulation oradministration method (e.g., a pump). However the compounds of theembodiments are administered to the subject, the amounts of compoundadministered and the route of administration chosen should be selectedto permit efficacious treatment of the disease condition.

The methods of the embodiments also include the use of a compound orcompounds as described herein together with one or more additionaltherapeutic agents for the treatment of disease conditions. Thus, forexample, the combination of active ingredients may be: (1) co-formulatedand administered or delivered simultaneously in a combined formulation;(2) delivered by alternation or in parallel as separate formulations; or(3) by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods described herein maycomprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used.

Diagnostic tests are contemplated as part of the methods describedherein. For example, a tissue biopsy sample may be taken from a subjectsuffering from an inflammatory condition, e.g., a p38-associated orcytokine-associated condition. The biopsy sample can be tested todetermine the level of p38 activity (or cytokine levels) present in thesample; the sample can then be contacted with a selected compound of theembodiments, and the p38 activity (or cytokine levels) measured todetermine whether the compound has a desired effect (e.g., inhibition ofp38 or cytokine activity with an EC₅₀ in the range of about 100 μM andabout 1000 μM, preferably about 50 μM to about 650 μM). Such a test maybe used to determine whether treatment with such a compound is likely tobe effective in that subject. Alternatively, the sample may be contactedwith a labeled compound (e.g., a fluorescently-labeled compound, or aradioactivity-labeled compound) and the sample then examined and thefluorescent or radioactive signal detected to determine the distributionof p38 in the tissue sample. Repeated biopsy samples taken during acourse of treatment may also be used to study the efficacy of thetreatment. Other diagnostic tests using the compounds described hereinwill be apparent to one of ordinary skill in the art in light of theteachings of this specification.

Thus, for example, an embodiment provides methods for determining thepresence, location, or quantity, or any combination thereof of p38protein in a cell or tissue sample. The methods include: a) contactingthe cell or tissue sample with a compound of the embodiments underconditions such that the compound can bind to at least one p38 MAPK; andb) determining the presence, location, or quantity, or any combinationthereof of the compound in the cell or tissue sample, therebydetermining the presence, location, or quantity, or any combinationthereof of the at least one p38 MAPK in the cell or tissue sample.Determining the presence, location, or quantity, or any combinationthereof of the compound in the cell or tissue sample may be conducted byany means that reveals the presence, location, or quantity, or anycombination thereof of the compound in the cell or tissue. For example,as described previously, radioactive or fluorescent labeling methods maybe used. Additional methods of determining the presence, location, orquantity, or any combination thereof of the compound will be apparent toa skilled artisan.

Another embodiment provides methods for determining: (1) whether acompound will be a useful therapeutic agent for treatment of a subjectsuffering from an inflammatory condition, or (2) the severity of diseaseor (3) the course of disease during treatment with a disease-modifyingagent. The methods include: a) obtaining a cell or tissue sample fromthe subject before, during and after termination of treatment with acompound as described herein or another disease-modifying agent; b)contacting the sample with the compound; and c) determining the amountof the compound that binds to the sample, wherein binding to p38 MAPK bythe compound is related to the amount of p38 MAPK in the sample.

Specific Examples of Diseases Contemplated to be Treated by theCompounds and Methods Described Herein COPD

Chronic obstructive pulmonary disease (COPD) is characterized by achronic inflammatory process in the lung that includes (1) increasednumber of inflammatory cells (neutrophils, macrophages and SD8⁺T cells)in the airways and parenchyma, (2) increased inflammatory cytokine andchemokine expression, and (3) increased number of proteases (elastases,cathepsins, and matrix metalloproteinases, MMPs). The production andaction of many of potential mediators of airway inflammation arebelieved to be dependent on the stress-induced MAPK or p38 kinasecascade. Several reports support the association pf p38 kinaseactivation with as plethora of pulmonary events: LPS- and TNF-α-inducedintercellular adhesion molecule-1 expression on pulmonary microvascularendothelial cells, MMP-9 activation, hypoxia-induced stimulation ofpulmonary arterial cells, hyperosmolarity-induced IL-8 expression inbronchial epithelial cells, and enhanced eosinophil trafficking andsurvival.

Trifilieff et al. (2005 Brit J Pharmacol 144:1002-10) reported thatCGH2466, a combined adenosine receptor antagonist, p38 MAPK andphosphodiesterase type 4 inhibitor showed potent in vitro and in vivoanti-inflammatory activities in diseases such as asthma and COPD.Underwood et al. (2000 Am J Physiol Lung Cell Mol Physiol 279:L895-L902)demonstrated that the potent and selective p38 MAPK inhibitor, SB239063,reduced proinflammatory cytokine production, including IL-1β, TNF-α,IL-6, and IL-8, which have been linked to airway fibrosis because oftheir ability to regulate fibroblast proliferation and matrixproduction; that leads to diminished neutrophil trafficking andactivation in the lung. Earlier, the same compound was found capable ofaltering responses associated with chronic fibrosis induced bybleomycin. This inhibitory activity was selective for the α and βisoforms of the p38. The compounds and methods described herein areuseful in the treatment of COPD.

Pulmonary Fibrosis

Pulmonary fibrosis also called idiopathic pulmonary fibrosis (IPF),interstitial diffuse pulmonary fibrosis, inflammatory pulmonaryfibrosis, or fibrosing alveolitis, is an inflammatory lung disorder anda heterogeneous group of conditions characterized by abnormal formationof fibrous tissue between alveoli caused by alveolitis comprising aninflammatory cellular infiltration into the alveolar septae withresulting fibrosis. The effects of IPF are chronic, progressive, andoften fatal. p38 MAPK activation has been demonstrated in the lung ofpatients with pulmonary fibrosis. A number of investigations aboutpulmonary fibrosis have indicated that sustained and augmentedexpression of some cytokines in the lung are relevant to recruitment ofinflammatory cells and accumulation of extracellular matrix componentsfollowed by remodeling of the lung architecture. In particular,proinflammatory cytokines such as TNF-α and interleukin IL-1β weredemonstrated to play major roles in the formation of pneumonitis andpulmonary fibrosis. In addition, profibrotic cytokines such as TGF-β andCTGF also play critical roles in the pathogenesis of pulmonary fibrosis.Matsuoka et al. (2002 Am J Physiol Lung Cell Mol Physiol 283:L103-L112)have demonstrated that a p38 inhibitor, FR-167653, ameliorates murinebleomycin-induced pulmonary fibrosis. Furthermore, pirfenidone(5-methyl-1-phenyl-2-(1H)-pyridone), a compound with combinedanti-inflammatory, antioxidant and antifibrotic effects was foundeffective in experimental models of pulmonary fibrosis as well as inclinical studies (Raghu et al. 1999 Am J Respir Crit Care Med159:1061-1069; Nagai et al. 2002 Intern Med 41:1118-1123; Gahl et al.2002 Mol Genet Metab 76:234-242; Azuma et al. 2002 Am J Respir Crit.Care Med 165:A729). The compounds and methods described herein areuseful in the treatment of pulmonary fibrosis, such as IPF.

Bronchiolitis Obliterans and Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans, and its correlating clinical conditionbronchiolitis obliterans syndrome, are characterized by obstruction ofthe pulmonary pathway via obliteration of pulmonary small airways. Inbronchiolitis obliterans, pathologic examination characteristicallyfinds lesions which obstruct or obliterate small airways in the lung.These lesions are granular fibromyxoid tissue and dense submucosal scartissue. Lesions progress from prolonged, abnormal, or aberrantinflammation of epithelial and epithelial-localized structures of thesmall airways, mediated by proinflammatory cytokines such as TNF-α andresult in excessive fibroproliferation. The obliteration of pulmonarysmall airways progressively leads to airflow obstruction, characterizedby progressive decline in forced expiratory volume in one second (FEV₁),and is often accompanied by recurring infections of the lowerrespiratory tract and colonization of pulmonary tissue by pathogenicmicroorganisms.

Bronchiolitis obliterans syndrome affects 50-60% of patients survivingfive years after lung transplantation surgery, and five year survivalafter onset of bronchiolitis obliterans syndrome is only 30-40%. Lungtransplantation patients experiencing bronchiolitis obliterans syndromeoften respond poorly to augmented immunosuppression. In patients withidiopathic pulmonary fibrosis, survival after bronchiolitis obliteranssyndrome is shorter than in patients with emphysema. The compounds andmethods described herein are useful in the treatment of bronchiolitisobliterans syndrome.

Chronic Allograft Fibrosis

Allograft failure is a profound concern in transplantation management.One of the primary causes of allograft failure is chronic allograftdysfunction. Hallmarks of chronic allograft dysfunction are chronicinflammation and chronic fibrosis, both of which are associated with theproduction of inflammatory cytokines and growth factors. Mediation ofinflammatory cytokine and growth factors, particularly resulting ininterruption of collagen and TGF production, is useful in the treatmentof chronic allograft fibrosis. The term “chronic allograft fibrosis” asused herein is intended to encompass both chronic inflammation andchronic fibrosis associated with chronic allograft fibrosis. Thecompounds and methods described herein are useful in the treatment ofchronic allograft fibrosis.

Renal Fibrosis

Irrespective of the nature of the initial insult, renal fibrosis isconsidered to be the common final pathway by which kidney diseaseprogresses to end-stage renal failure. Stambe et al. (2004 J Am SocNephrol 15:370-379) tested an inhibitor of the active (phosphorylated)form of p38, NPC 31169, developed by Scios Inc. (San Francisco, Calif.)in a rat model of renal fibrosis, and reported a significant reductionin renal fibrosis assessed by interstitial volume, collagen IVdeposition, and connective tissue growth mRNA levels. The compounds andmethods described herein are useful in the treatment of renal fibrosis.

Leiomyoma

Uterine leiomyomas or fibroids are the most common pelvic tumors inwomen with no known long-term effective drug therapies available.Leiomyomas are characterized by increased cell proliferation and tissuefibrosis. Pirfenidone was tested on cell proliferation and collagenexpression in cultured myometrial and leiomyoma smooth muscle cells, andwas found to be an effective inhibitor of myometrial and leiomyoma cellproliferation (Lee et al. 1998 J Clin Endocrinol Metab 83:219-223). Thecompounds and methods described herein are useful in the treatment ofleiomyomas.

Endomyocardial Fibrosis

Endomyocardial fibrosis (EMF) is a disorder characterized by thedevelopment of restrictive cardiomyopathy. EMF is sometimes consideredpart of a spectrum of a single disease process that includes Lófflerendocarditis (nontropical eosinophilic endomyocardial fibrosis orfibroplastic parietal endocarditis with eosinophilia). In EMF, theunderlying process produces patchy fibrosis of the endocardial surfaceof the heart, leading to reduced compliance and, ultimately, restrictivephysiology as the endomyocardial surface becomes more generallyinvolved. Endocardial fibrosis principally involves the inflow tracts ofthe right and left ventricles and may affect the atrioventricularvalves, leading to tricuspid and mitral regurgitation. MAPK activationwas shown to contribute to arrhythmogenic atrial structural remodelingin EMF. The compounds and methods described herein are useful in thetreatment and/or prevention of endomyocardial fibrosis.

Other Inflammatory Diseases

Many autoimmune diseases and diseases associated with chronicinflammation, as well as acute responses, have been linked to activationof p38 MAP kinase and overexpression or dysregulation of inflammatorycytokines. These diseases include, but are not limited to: rheumatoidarthritis; rheumatoid spondylitis; osteoarthritis; gout, other arthriticconditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis;toxic shock syndrome; asthma; adult respiratory distress syndrome;chronic obstructive pulmonary disease; chronic pulmonary inflammation;inflammatory bowel disease; Crohn's disease; psoriasis; eczema;ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute andchronic renal disease; irritable bowel syndrome; pyresis; restenosis;cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer'sdisease; Huntington's disease; Parkinson's disease; acute and chronicpain; allergic rhinitis; allergic conjunctivitis; chronic heart failure;acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lymedisease; Reiter's syndrome; acute synoviitis; muscle degeneration,bursitis; tendonitis; tenosynovitis; herniated, ruptures, or prolapsedintervertebral disk syndrome; osteopetrosis; thrombosis; cancer;restenosis; silicosis; pulmonary sarcosis; bone resorption diseases,such as osteoporosis; graft-versus-host reaction; and auto-immunediseases, such as Multiple Sclerosis, lupus and fibromyalgia; AIDS andother viral diseases such as Herpes Zoster, Herpes Simplex I or II,influenza virus and cytomegalovirus; and diabetes mellitus.

Many studies have shown that reducing the activity of p38 MAP kinase,its upstream activators or its downstream effectors, either throughgenetic or chemical means, blunts the inflammatory response and preventsor minimizes tissue damage (see, e.g., English, et al. 2002 TrendsPharmacol Sci 23:40-45; and Dong et al. 2002 Annu Rev Immunol 20:55-72).Thus, inhibitors of p38 activity, which also inhibit excess orunregulated cytokine production and may inhibit more than a singlepro-inflammatory cytokine, may be useful as anti-inflammatory agents andtherapeutics. Furthermore, the large number of diseases associated withp38 MAP kinase-associated inflammatory responses indicates that there isa need for effective methods for treating these conditions.

Cardiovascular disease. Inflammation and leukocyteactivation/infiltration play a major role in the initiation andprogression of cardiovascular diseases including atherosclerosis andheart failure. Acute p38 mitogen-activated protein kinase (MAPK) pathwayinhibition attenuates tissue damage and leukocyte accumulation inmyocardial ischemia/reperfusion injury. The compounds and methodsdescribed herein are useful for treating cardiovascular disease.

Multiple sclerosis. Inflammation in the central nervous system occurs indiseases such as multiple sclerosis and leads to axon dysfunction anddestruction. Both in vitro and in vivo observations have shown animportant role for nitric oxide (NO) in mediating inflammatoryaxonopathy. p38 MAP kinase is activated by NO exposure and inhibition ofp38 signalling was shown to lead to neuronal and axonal survivaleffects. OCM and IGF-1 reduced p38 activation in NO-exposed corticalneurons and improved axon survival in cultures exposed to NO, a processdependent on mitogen-activated protein kinase/extracellularsignal-related kinase signalling. The compounds and methods describedherein are useful for treating multiple sclerosis.

Primary graft nonfunction. Nonspecific inflammation is associated withprimary graft nonfunction (PNF). Inflammatory islet damage is mediatedat least partially by pro-inflammatory cytokines, such as interleukin-1β(IL-1β) and tumor necrosis factor-α (TNF-α) produced by resident isletmacrophages. The p38 pathway is known to be involved in cytokineproduction in the cells of the monocyte-macrophage lineage. Inhibitionof the p38 pathway by a chemical p38 inhibitor, SB203580, suppressesIL-1β and TNF-α production in human islets exposed to lipopolysaccharide(LPS) and/or inflammatory cytokines. Although IL-1β is predominantlyproduced by resident macrophages, ductal cells and islet vascularendothelial cells were found to be another cellular source of IL-1β inisolated human islets. SB203580 also inhibited the expression ofinducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) inthe treated islets. Furthermore, human islets treated with SB203580 for1 h prior to transplantation showed significantly improved graftfunction. The compounds and methods described herein are useful forimproving graft survival in clinical islet transplantation.

Acute renal injury. Cisplatin is an important chemotherapeutic agent butcan cause acute renal injury. Part of this acute renal injury ismediated through tumor necrosis factor-α (TNF-α). Cisplatin activatesp38 MAPK and induces apoptosis in cancer cells. p38 MAPK activationleads to increased production of TNF-α in ischemic injury and inmacrophages. In vitro, cisplatin caused a dose dependent activation ofp38 MAPK in proximal tubule cells. Inhibition of p38 MAPK activation ledto inhibition of TNF-α production. In vivo, mice treated with a singledose of cisplatin developed severe renal dysfunction, which wasaccompanied by an increase in kidney p38 MAPK activity and an increasein infiltrating leukocytes. However, animals treated with a p38 MAPKinhibitor SKF86002 along with cisplatin showed less renal dysfunction,less severe histologic damage and fewer leukocytes compared withcisplatin+vehicle treated animals. The compounds and methods describedherein are useful for preventing acute renal injury.

Periodontitis. The proinflammatory mediator bradykinin (BK) stimulatesinterleukin-8 (IL-8) production in human gingival fibroblasts in vitroand plays an important role in the pathogenesis of various inflammatorydiseases including periodontitis. The specific p38 mitogen-activatedprotein kinase (MAPK) inhibitor SB 203580 reduced IL-8 productionstimulated by the combination of BK and IL-1β as well as theIL-1β-stimulated IL-8 production. The compounds and methods describedherein are useful for treating or preventing periodontitis.

Pharmaceutical Compositions

While it is possible for the compounds described herein to beadministered alone, it may be preferable to formulate the compounds aspharmaceutical compositions. As such, in yet another aspect,pharmaceutical compositions useful in the methods of the invention areprovided. More particularly, the pharmaceutical compositions describedherein may be useful, inter alia, for treating or preventinginflammatory conditions, e.g., conditions associated with p38 activityor cytokine activity or any combination thereof. A pharmaceuticalcomposition is any composition that may be administered in vitro or invivo or both to a subject in order to treat or ameliorate a condition.In a preferred embodiment, a pharmaceutical composition may beadministered in vivo. A subject may include one or more cells ortissues, or organisms. A preferred subject is a mammal. A mammalincludes any mammal, such as by way of non-limiting example, cattle,pigs, sheep, goats, horses, camels, buffalo, cats, dogs, rats, mice, andhumans. A highly preferred subject mammal is a human.

In an embodiment, the pharmaceutical compositions may be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. The pharmaceutical compositionsshould generally be formulated to achieve a physiologically compatiblepH, and may range from a pH of about 3 to a pH of about 11, preferablyabout pH 3 to about pH 7, depending on the formulation and route ofadministration. In alternative embodiments, it may be preferred that thepH is adjusted to a range from about pH 5.0 to about pH 8. Moreparticularly, the pharmaceutical compositions may comprise atherapeutically or prophylactically effective amount of at least onecompound as described herein, together with one or more pharmaceuticallyacceptable excipients. Optionally, the pharmaceutical compositions maycomprise a combination of the compounds described herein, or may includea second active ingredient useful in the treatment or prevention ofbacterial infection (e.g., anti-bacterial or anti-microbial agents).

Formulations, e.g., for parenteral or oral administration, are mosttypically solids, liquid solutions, emulsions or suspensions, whileinhalable formulations for pulmonary administration are generallyliquids or powders, with powder formulations being generally preferred.A preferred pharmaceutical composition may also be formulated as alyophilized solid that is reconstituted with a physiologicallycompatible solvent prior to administration. Alternative pharmaceuticalcompositions may be formulated as syrups, creams, ointments, tablets,and the like.

The term “pharmaceutically acceptable excipient” refers to an excipientfor administration of a pharmaceutical agent, such as the compoundsdescribed herein. The term refers to any pharmaceutical excipient thatmay be administered without undue toxicity.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there exists awide variety of suitable formulations of pharmaceutical compositions(see, e.g., Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA;carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid; liquids such as oils, water,saline, glycerol and ethanol; wetting or emulsifying agents; pHbuffering substances; and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions described herein may be formulated inany form suitable for the intended method of administration. Whenintended for oral use for example, tablets, troches, lozenges, aqueousor oil suspensions, non-aqueous solutions, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, syrups or elixirs may be prepared. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as cross-linked povidone, maizestarch, or alginic acid; binding agents, such as povidone, starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc,

Tablets may be uncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions may be formulated assuspensions comprising a compound of the embodiments in admixture withat least one pharmaceutically acceptable excipient suitable for themanufacture of a suspension.

In yet another embodiment, pharmaceutical compositions may be formulatedas dispersible powders and granules suitable for preparation of asuspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wettingagents such as a naturally occurring phosphatide (e.g., lecithin), acondensation product of an alkylene oxide with a fatty acid (e.g.,polyoxyethylene stearate), a condensation product of ethylene oxide witha long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate); polysaccharides and polysaccharide-like compounds (e.g.dextran sulfate); glycosaminoglycans and glycosaminoglycan-likecompounds (e.g., hyaluronic acid); and thickening agents, such ascarbomer, beeswax, hard paraffin or cetyl alcohol. The suspensions mayalso contain one or more preservatives such as acetic acid, methyland/or n-propyl p-hydroxy-benzoate; one or more coloring agents; one ormore flavoring agents; and one or more sweetening agents such as sucroseor saccharin.

The pharmaceutical compositions may also be in the form of oil-in wateremulsions. The oily phase may be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth; naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids; hexitol anhydrides, such as sorbitan monooleate; and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan monooleate. The emulsion may also containsweetening and flavoring agents. Syrups and elixirs may be formulatedwith sweetening agents, such as glycerol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, a flavoringor a coloring agent.

Additionally, the pharmaceutical compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may likewise be used in the preparation of injectables.

To obtain a stable water-soluble dose form of a pharmaceuticalcomposition, a pharmaceutically acceptable salt of a compound describedherein may be dissolved in an aqueous solution of an organic orinorganic acid, such as 0.3 M solution of succinic acid, or morepreferably, citric acid. If a soluble salt form is not available, thecompound may be dissolved in a suitable co-solvent or combination ofco-solvents. Examples of suitable co-solvents include alcohol, propyleneglycol, polyethylene glycol 300, polysorbate 80, glycerin and the likein concentrations ranging from about 0 to about 60% of the total volume.In one embodiment, the active compound is dissolved in DMSO and dilutedwith water.

The pharmaceutical composition may also be in the form of a solution ofa salt form of the active ingredient in an appropriate aqueous vehicle,such as water or isotonic saline or dextrose solution. Also contemplatedare compounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, etc.

In a preferred embodiment, the compounds described herein may beformulated for oral administration in a lipid-based formulation suitablefor low solubility compounds. Lipid-based formulations can generallyenhance the oral bioavailability of such compounds.

As such, a preferred pharmaceutical composition comprises atherapeutically or prophylactically effective amount of a compounddescribed herein, together with at least one pharmaceutically acceptableexcipient selected from the group consisting of—medium chain fatty acidsor propylene glycol esters thereof (e.g., propylene glycol esters ofedible fatty acids such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants such as polyoxyl 40 hydrogenatedcastor oil.

In an alternative preferred embodiment, cyclodextrins may be added asaqueous solubility enhancers. Preferred cyclodextrins includehydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosylderivatives of α-, β-, and γ-cyclodextrin. A particularly preferredcyclodextrin solubility enhancer is hydroxypropyl-o-cyclodextrin (BPBC),which may be added to any of the above-described compositions to furtherimprove the aqueous solubility characteristics of the compounds of theembodiments. In one embodiment, the composition comprises about 0.1% toabout 20% hydroxypropyl-o-cyclodextrin, more preferably about 1% toabout 15% hydroxypropyl-o-cyclodextrin, and even more preferably fromabout 2.5% to about 10% hydroxypropyl-o-cyclodextrin. The amount ofsolubility enhancer employed will depend on the amount of the compoundof the embodiments in the composition.

A pharmaceutical composition contains a total amount of the activeingredient(s) sufficient to achieve an intended therapeutic effect. Morespecifically, in some embodiments, the pharmaceutical compositioncontains a therapeutically effective amount (e.g., an amount of anSAPK-modulating compound that is effective in the prevention ortreatment of the symptoms of an inflammatory disease or condition,wherein the compound exhibits an EC₅₀ in the range of about 1 μM toabout 1000 μM, preferably about 50 μM to about 650 μM, for inhibition ofat least one p38 MAPK). The total amounts of the compound that may becombined with the carrier materials to produce a unitary dosing formwill vary depending upon the host treated and the particular mode ofadministration. Preferably, the compositions are formulated so that adose of between 0.01 to 100 mg/kg body weight/day of an SAPK-modulatingcompound is administered to a patient receiving the compositions.

Example 1

Compounds are screened for the ability to inhibit ATF2 phosphorylationby p38 MAP kinase in vitro. The ability of compounds to inhibit ATF2phosphorylation in this in vitro assay is correlated with the inhibitionof p38 MAP kinase and TNFα expression in vivo, and is therefore anindicator of potential in vivo therapeutic activity (Raingeaud, J. etal. 1995 J. Biol. Chem. 270:7420-7426; Brinkman, M. N., et al. 1999 J.Biol. Chem. 274:30882-30886; and Fuchs, S. Y. et al. J. Biol. Chem.275:12560-12564, 2000).

All kinases and the substrate ATF2 are acquired from Upstate(Charlottesville, Va.). p38 MAP Kinases are recombinant humanfull-length proteins with an amino-terminal GST fusion, expressed in andpurified from E. coli. ATF2 is a GST fusion protein containing aminoacids 19-96 of human ATF2 expressed in E. coli. All proteins arealiquoted and stored at −80° C.

p38 MAP kinase assays are performed using an assay buffer containing 25mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM DTT, 20 mM β-glycerophosphate, 0.1mM Na₃VO₄, 40 μM ATP and 1.25 μM of ATF2, together with 6 ng of p38αprotein, 12 ng p38β protein, 1.5 ng p38γ, or 0.4 ng JNK2α2. Compoundsare serially diluted in DMSO and 2 μL of test compound at variousconcentrations is used. The vehicle control receives DMSO only.

Test compounds are pre-incubated with 20 μl of enzyme in kinase buffer(25 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM DTT, 20 mM β-glycerophosphateand 0.1 mM Na₃VO₄) at room temperature for 15 minutes. Reactions areinitiated by addition of 30 μl substrate solution to yield a finalconcentration of 40 μM ATP and 1.25 μM ATF2 in kinase buffer. Thereactions are incubated for 30 minutes at 37° C. and terminated by theaddition of 18 μl of 200 mM EDTA. An ELISA method is used to measure thephosphorylation of ATF2 at Thr 69. High binding 96-well plates arecoated with 50 μl of kinase reaction for 1 hour at 37° C. The coatedplates are washed with 200 μl washing buffer (25 mM Tris HCl, pH 8.3,192 mM glycine, 0.1% SDS and 0.05% Tween-20) three times. The plates arethen washed three times with SuperBlock in TBS (Pierce, 37535). Afterblocking, plates are incubated with 50 μl of rabbit anti-phospho-ATF2antibody (Cell Signaling, 9221L, 1:500) for 30 minutes at 37° C.

Plates are washed three times with washing buffer prior to incubationwith 50 μl HRP-conjugated goat anti-rabbit antibody (Cell Signaling,7074, 1:500) for 30 minutes at 37° C. Plates are then washed three timeswith washing buffer before incubation with 50 μl of Ultra TMB-ELISA(Pierce, 34028) for 8 minutes at room temperature. Finally, 50 μl ofphosphoric acid (1 M) is added to stop reactions and plate absorbance isread at 450 nm on a SpectraMax 250 plate reader.

The compounds inhibit the phosphorylation of ATF2 in this in vitroassay. Preferred compounds exhibit EC₅₀ values of between about 1 μM andabout 1000 μM, preferably about 50 μM to about 650 μM.

Example 2

Compounds are screened for the ability to inhibit TNFα release fromTHP-1 cells stimulated with lipopolysaccharide (LPS) in vitro. Theability of compounds to inhibit TNFα release in this in vitro assay iscorrelated with the inhibition of p38 activity and TNFα, and istherefore an indicator of potential in vivo therapeutic activity (Lee J.C. et al. 1993 Ann. NY. Acad. Sci. 696:149-170; and 1994 Nature372:739-746).

THP-I cells from ATCC (TIB202) are maintained at 37° C., 5% CO₂ in RPMI1640 media (MediaTech, Herndon, Va.) containing 4.5 g/L glucose,supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and50 μM β-mercaptoethanol.

Test compounds are initially dissolved in RPMI media with 1% DMSO (v/v).Compounds are then serially diluted in RPMI media for all subsequentdilutions. The assay is performed under sterile conditions. THP-1 cellsat a culture density of 6-8×10⁵ cells/ml are collected and resuspendedin the RPMI media at 10⁶ cells/ml. 100 μl of resuspended cells are addedto each well, which contain 100 μl of a test compound. Test compoundsare prepared at twice the final concentration. Final DMSO concentrationis no more than 0.5% (v/v). Cells are preincubated with compound for 60minutes at 37° C., 5% CO₂ prior to stimulation with lipopolysaccharide(LPS) (Sigma L-2880, 4 mg/ml stock in PBS). The final LPS concentrationin each well is 10 or 30 μg/ml for TNFα and IL-1β release, respectively.Unstimulated control cell suspensions receive PBS vehicle only. Cellmixtures are incubated for 18 or 48 hours for TNFα and IL-1β release,respectively. 150 μl of supernatants are taken and transferred to afresh plate and stored at −20° C. until farther analysis. TNFα and IL-1βlevels are measured using ELISA kits. A Luminescence is used as theplate reader. Analysis is performed by non-linear regression to generatea dose response curve. The calculated EC₅₀ value is the concentration ofthe test compound that causes a 50% decrease in TNFα or IL-1β levels.

Compounds inhibit the release of TNFα, IL-1β or both TNFα, and IL-1β inthis in vitro assay. Preferred compounds exhibit EC₅₀ values for TNFαand/or IL-1β of between about 1 μM and about 1000 μM, preferably about50 μM to about 650 μM. Data are provided in Table 2 below.

TABLE 2

TNF No.¹ X₃ R₂ R₄ Z EC₅₀ (μM)² Toxicity³ 13 —H —CH₃ —H O A ≧1 mM 7 —H —H—H O B D 9 —OH —H —H O C D 11 —F —H —H O B D 8 —H —CF₃ —H O B D 12 —F—CF₃ —H O C D 5 —OH —CH₃ —H O A ≧1 mM 1 —H —CH₂OH —H O B D 2 —H —COOH —HO C D 3 —H -glucuronide —H O C D 6 —H —CH₂OCH₃ —H O A ≧1 mM 4 —H —CH₃—OH O A ≧1 mM 14 —F —CH₃ —H O B D 15 —OCH₃ —CF₃ —H O C D 16 —COCH₃ —H —HO C D 10 —OH —CF₃ —H O A D 17 —H -phenyl —H O C D 18 —H —CH₃ —H S C D 25See note CH₃ —H O B B 4 26 —H —Br —H O A B 27 —OCH₃ —Br —H O A A 28 —OH—CH₂F —H O C D 29 —OH —CHF₂ —H O C D 30 —OH —Br —H O A A 31 —OCH₃ —CH₂F—H O A A 32 —OCH₃ —CHF₂ —H O A A 33 —H See note 5 —H O C D 24 —H CO₂CH₃—H O C D ¹Compound number as shown in Table 1 ²A: ≦2,000; B: >2,000; C:Inconclusive (e.g., no data or no activity observed) ³D: Inconclusive(e.g., no data or no toxicity observed) ⁴Compound 25 is as depicted inTable 1, namely the aryl group attached to a 2-pyridone nitrogen is anN-methylpyridinium moiety ⁵Compound 33 is as depicted in Table 1, namelya bromoaryl group fused to the 5 and 6 positions of a 2-pyridone ring

Example 3

Compounds are screened for the ability to inhibit TNFα release fromprimary human peripheral blood mononuclear cells (PBMC) stimulated withlipopolysaccharide (LPS) in vitro. The ability of compounds to inhibitTNFα release in this in vitro assay is correlated with the inhibition ofp38 activity and is therefore an indicator of potential in vivotherapeutic activity (2002 Osteoarthritis & Cartilage 10:961-967; andLaufer, S. A. and Wagner, G. K. 2002 J. Med. Chem. 45: 2733-2740).

Human peripheral blood mononuclear cells (PBMC) are isolated bydifferential centrifugation through a Ficoll-HyPaque density gradientfrom pooled serum of 3-8 individual blood donors. Isolated PBMC containapproximately 10% CD-14 positive monocytes, 90% lymphocytes and <1%granulocytes and platelets. PBMC (10⁶/ml) are cultured in polystyreneplates and stimulated with lipopolysaccharide (LPS; 50 ng/ml; Sigma, St.Louis, Mo.) in the presence and absence of the test compound in serialdilutions, in duplicate, for 24 hr at 37° C. in GIBCO™ RPM1 medium(Invitrogen, Carlsbad, Calif.) without serum. The TNFα level in cellsupernatants is determined by ELISA using a commercially available kit(MDS Panlabs #309700).

Preferred compounds inhibit the release of TNFα in this assay with anEC₅₀ value of between about 1 μM and about 1000 μM, preferably about 50μM to about 650 μM.

Example 4

Compounds are screened for the ability to inhibit the release of TNFα inan in vivo animal model (See, e.g., Griswold D. E. et al. 1993 DrugsExp. Clin. Res. 19:243-248; Badger, A. M. et al. 1996 J. Pharmacol. Exp.Ther. 279:1453-1461; Dong, C. et al. 2002 Annu. Rev. Immunol. 20:55-72(and references cited therein); Ono, K. and Han, J. 2000 CellularSignalling 12:1-13 (and references cited therein); and Griffiths, J. B.et al. 1999 Curr. Rheumatol. Rep. 1:139-148).

Without being bound by any particular theory, it is believed thatinhibition of TNFα in this model is due to inhibition of p38 MAP kinaseby the compound.

Male Sprague-Dawley rats (0.2-0.35 kg) are randomly divided into groupsof six or more and are dosed intravenously by infusion or bolusinjection, or are dosed orally with test compounds in a suitableformulation in each case. Thirty minutes following end of infusion orbolus injection, and 1-2 hr following oral administration,lipopolysaccharide E. coli/0127:B8 (0.8 mg/kg) is administered IV. Bloodsamples are collected 1.5 hours post-treatment with LPS. Serum TNFαlevels are determined using the ELISA kit from Biosource (KRC3011C) andcompared to that from vehicle-treated control.

Preferred compounds inhibit the release of TNFα in this in vivo assay.Preferred compounds exhibit an ED₅₀ value of less than 500 mg/kg,preferably less than 400 mg/kg, preferably less than 200 mg/kg,preferably less than 100 mg/kg, more preferably, less than 50 mg/kg,more preferably, less than 40 mg/kg, more preferably, less than 30mg/kg, more preferably, less than 20 mg/kg, more preferably, less than10 mg/kg.

The methods of determining the EC₅₀ of the inhibition of p38 by acompound include any methods known in the art that allow thequantitative detection of any of the downstream substrates of p38 MAPKas described above. Therefore, these methods additionally include butlimited to detection of expression of genes known to be regulated by p38either individually, or by gene arrays.

Example 5

The following methods can be used for (1) a kinase assay fordetermination of EC₅₀, (2) a non-radiometric kinase assay fordetermination of EC₅₀, (3) modulation of induction of TNFα expression,(4) a test for cell toxicity, and (5) an assay to test the effect ofcompounds on collagen production.

Kinase Assay

The activity of the P38 kinase isoforms P38γ and P38α is determined byphosphorylation of ATF-2 in presence of ³²P-γ-ATP. The incorporation of³²P into ATF-2 in the presence or absence of inhibitors is determined.Pirfenidone and its different derivatives are tested for inhibition ofP38γ and P38α kinase activity in this biochemical assay. The compoundsare solubilized in water or DMSO and tested at different concentrationsfrom 0 to 10 mM using the appropriate solvent for dilutions and asvehicle control. The enzymes P38γ and P38α are obtained as activated andpurified recombinant protein (Upstate, Charlottesville, Va.). Theactivated enzyme is used at 24.8 nM in the final reaction. The enzymesare diluted prior to the reaction in the following buffer (1M HEPES, pH7.4, 500 mM DTT, 1% Triton X-100 and 10 mg/ml BSA). The reaction isperformed in the following solution that is prepared as a two fold stocksolution (1M HEPES, pH 7.4, 500 mM DTT and 1% Triton X-100) andnon-radioactive ATP is present in the reaction at 6.25 μM ATP (CellSignaling, Beverly, Mass.). To determine the phosphorylation of ATF-2,γ-[³²P]-ATP 3000 Ci/mmol is added to each reaction at a concentration of7.5 μM. ATF-2 (Cell Signaling, Beverly, Mass.) as a kinase substrate isused at 3 μM. As a first step in assembling the enzyme reaction,activated kinase and inhibitor or the appropriate vehicle control areadded to reaction buffer and incubated for 30 min at room temperature.The kinase reaction is initiated by the addition of ATF-2 and ATPmixture. The final volume for each reaction is 20 μA and performed atroom temperature for 30 minutes. After the 30 minutes of incubation 80ul of Laemmlie buffer is added. Subsequently 20% of the reaction isseparated by SDS-Page (BioRad, Hercules, Calif.) under reducingconditions. After electrophoresis, the gel is exposed to aphosphorimager plate and analyzed using a phosphoimager (Storm System,Amersham Biosciences, Piscataway, N.J.). The signal obtained isquantified after background correction and calculated as percentinhibition using the uninhibited kinase activity with the vehiclecontrol as 0% inhibition. The kinase activity, in the presence ofdifferent inhibitor concentrations, is plotted using Kaleidagraph(Synergy Software, Reading, Pa.) to determine the EC₅₀ for each compoundand tested P38 kinase.

Non-Radiometric Kinase Assay

An alternate, non-radiometric kinase assay was also employed to definethe EC50 for inhibition of P38. In this assay, p38 kinase transfers aphosphate from ATP to an EGF-R peptide substrate, resulting in theformation of phosphorylated EGF-R peptide with the concomitantconversion of ATP to ADP. In an uncoupled reaction, p38 also hydrolyzesATP at a slower rate in the absence of peptide substrate (Fox et al,FEBS Lett 1999), which contributes slightly to ATP consumption. Thus,the amount of ATP consumed is directly proportional to p38 activity. Atthe end of a kinase reaction, the amount of ATP remaining is determinedusing Kinase-Glo Plus Luminescent Kinase Assay (Promega, Inc., Madison,Wis.). These reagents use residual ATP to support the ATP-dependentenzymatic conversion of beetle luciferin to oxyluciferin with theconcomitant production of light, which is detected by a luminometer.

Kinase reactions are conducted by mixing compound (diluted in DMSO andassay buffer) with either p38α or p38γ, and EGF-R peptide substrate(AnaSpec, Inc., San Jose, Calif.) in assay buffer. Reactions are theninitiated by the addition of ATP and allowed to run for 45 minutes atroom temperature. Final buffer conditions are: 20 mM HEPES (pH 7.4), 2mM DTT, 0.1% Triton-X-100, 10 mM MgCl₂, 10% glycerol, 12.5 mM p38α orp38γ (Upstate, Charlottesville, Va.), 50 μM EGF-R peptide substrate, and10 μM ATP. The final assay volume is 10 μL. A control reaction isperformed in the absence of compound. Additional control reactions thatomit p38 are performed at every compound concentration. All reactionsare performed in triplicate.

Forty-five minutes after initiation of the kinase reaction, the reactionis quenched by the addition of 10 μL of Kinase-Glo Plus assay reagent.The luciferase reaction is allowed to equilibrate for 15 minutes priorto being read on an Envision Multilabel Plate Reader (Perkin Elmer Lifeand Analytical Sciences, Boston, Mass.). Data are plotted as luminescentsignal versus log compound concentration in KaleidaGraph (SynergySoftware, Reading, Pa.). EC₅₀ values are determined by fitting the datato 4-paramater binding equation using a fixed upper bound that isdetermined from control reactions in the absence of p38 (as luminescenceis inversely related to kinase activity).

Inhibition of TNFα Induction

THP-1 (ATCC, Rockville, Md.) is grown under regular tissue cultureconditions as recommended by ATCC. 18 hours prior to the experiment,cells were plated in a 96 well format in regular culture mediacontaining 1% serum and 0.25 ml culture volume at a density of 500,000cells per well. The compound is added to each well in triplicates andthe appropriate solvent control is included in each assay. The p38inhibitor SB203850 at 1 mM/ml (Upstate, Waltham, Mass.) is included as apositive control in each assay. For the induction of TNFα expression, 1μg/ml LPS is added to each well 30 minutes post compound addition.Following a 4 hour incubation under tissue culture conditions the cellsare sedimented by centrifugation (10 min, 1000 rpm, Beckman table topcentrifuge) and a fraction of the cell free supernatant is collected andused in a tenfold dilution for the quantification in the TNFα specificELISA (R&D Systems, Minneapolis, Minn.). The TNFα ELISA is performedaccording to the directions provided by the manufacturer. The TNFα isdetected in pg/ml and plotted as fractional activity normalized to theTNFα expression in the solvent control.

Compound Toxicity Testing in a Cell Based Assay

The release of LDH as result of a disrupted cell membrane is applied asa measure of cell toxicity. LDH is detected by its enzymatic activityusing a commercially available diagnostic kit (Roche Diagnostics, Cat# 1644 793). THP-1 cells are used for determination of cell toxicity forconsistency with the induced TNFα expression in the previous experiment.As previously described for testing of inhibition of TNFα induction,cells are cultured in a 96 well format under 1% serum and regular tissueculture conditions. The compounds are added at different concentrationsin triplicate. The appropriate solvent control is used in each assay.After compound addition the cells are cultured 18 hours under regulartissue culture conditions. After this incubation period, the positivecontrol is initiated by adding Triton-X-100 (2% v/v) to untreated cellsand incubated for an additional 10 minutes for complete cell lysis.Subsequently the cells are sedimented by centrifugation and a fractionof the supernatant removed and analyzed for LDH enzyme activityaccording to the manufacturer's instructions. The data is typicallyreported as % cell toxicity normalized to the Triton-X-100 lysed cellsas 100% cell toxicity.

Toxicity data are also obtained using a commercially available ATP assay(Molecular Probes' ATP Determination Kit A22066, available fromInvitrogen) and/or using a MTT assay. Both the ATP and the MTT assaysmeasure metabolic competence of the cell. The MTT assay measures theability of the cell to reduce a marker substrate, which is related tometabolic competence (i.e. viability). The ATP assay measures thecellular ATP concentration in the presence and absence of compound.Toxic compounds lead to reduced metabolic activity which leads to areduction in ATP concentration.

Assay for Effect of Compounds on Collagen Production

HFL-1 Cells (ATCC, Rockville, Md.) were grown under regular tissueculture conditions in complete media containing 10% fetal bovine serum(FBS; Mediatech, Inc., Herndon, Va.). Cells in early passage were platedin 6 well plates. When the cells reached confluence, the media wasremoved, cells washed with PBS, and the cells were kept overnight incomplete media containing 0.1% FBS. The media was then replaced withfresh media plus 0.1% FCS, 10 μM L-Proline (EMD Chemicals, Gibbstown,N.J.), 20 μg/mL ascorbic acid (EMD Chemicals, Gibbstown, N.J.).Compounds were added to triplicate wells to a final concentration of 1mM from 100× stock solutions in DMSO. One hour after the addition ofcompound, the cells were treated with TGF-β1 (Sigma-Aldrich, St. Louis,Mo.) to a final concentration of 10 ng/mL (25 ng total). Three daysafter addition of TGF-β, the media was removed, cells were washed withPBS and then lysed. The total collagen content of lysed cells wasassessed with a dye-based collagen assay (Sircol Collagen Assay,Newtownabbey, Northern Ireland) and a μQuant plate-basedspectrophotometer (BioTek Instruments, Inc., Winooski, Vt.) withappropriate standard curves. The dynamic range of the assay is definedby cells that were mock treated (1% DMSO without compound) in thepresence and absence of TGF-β. Data are reported in Table 3 as thepercent inhibition of TGF-β-induced collagen as determined in thefollowing equation:

% inhibition=100*[(collagen, mock/+TGF-β)−(collagen,treated/+TGF-β)]/[(collagen, mock/+TGF-β)−(collagen, mock/−TGF-β)]

TABLE 3 % Inhibition of TGF-β Stimulated Compound No. Collagen Synthesis8 48 14 23 15 49 26 58 30 69

Example 6

Preparation of 1-(4-hydroxyphenyl)-5-(trifloromethyl)-2-pyridone(Compound 10): A mixture of 5-(trifloromethyl)-2(1H)-pyridone (815.5 mg,5 mmol), 4-iodoanisole (2.34 g, 10 mmol), CuI (952 mg, 5 mmol), K₂CO₃(691 mg, 5 mmol) and DMF (5 ml) was heated at 135° C. overnight. Thereaction mixture was diluted with 10% ammonia (15 ml) and extracted withethyl acetate. The organic extract was washed with saturated sodiumchloride, dried over magnesium sulfate and evaporated. Columnchromatography purification (30% ethyl acetate-hexane) afforded 526 mg(39.2%) of 1-(4-methoxyphenyl)-5-(trifloromethyl)-2-pyridone. Thiscompound (268.2 mg, 1 mmol) was treated with 1M BBr₃ solution indichloromethane (DCM, 2 ml) in DCM (5 ml) for 2 hours at 0° C. Reactionmixture was diluted with DCM and washed 3 times with water. Organicphase was dried over sodium sulfate and evaporated. The residue wasseparated by column chromatography (20% ethyl acetate-DCM) to afford thetitle compound as a off-white solid, 226 mg (89%). The ¹H NMR spectrawas consistent with the structure of Compound 10.

Example 7

Preparation of 1-phenyl-5-acetyl-2-pyridone (Compound 16):2-methoxy-5-acetyl pyridine (1.51 g, 10 mmol) was treated with 6N HCl at100° C. for 5 hours. The reaction mixture was neutralized with sodiumhydroxide to pH 7 and then extracted several times with DCM. Organiclayer was dried over sodium sulfate, evaporated and the residue wascrystallized from ethyl acetate to give 5-acetyl-2(1H)-pyridone as awhite solid, 1.06 g (78%). This compound (685.7 mg, 5 mmol) was reactedwith iodobenzene (0.84 ml, 7.5 mmol) in the presence of CuI (95 mg, 0.5mmol) and K₂CO₃ (691 mg, 5 mmol) in DMF (5 ml) at 135° C. overnight. Thereaction mixture was diluted with 10% ammonia (15 ml) and extracted withethyl acetate. The organic extract was washed with saturated sodiumchloride, dried over magnesium sulfate and evaporated. Columnchromatography (10% ethyl acetate-DCM) afforded 407 mg (38%) of thetarget compound as a white solid. The ¹H NMR spectra was consistent withthe structure of Compound 16.

Example 8

Preparation of 1-(4-pyridinyl)-5-methyl-2-pyridone (Compound 22):Compound 22 was synthesized by condensation of 5-methyl-2(1H)-pyridone(327.4 mg, 3 mmol) with 4-bromopyridine hydrochloride (778 mg, 4 mmol)in the presence of CuI (60 mg, 0.3 mmol) and K₂CO₃ (1.36 g, 10 mmol) inDMF (3 ml) at 135° C. overnight. The reaction mixture was diluted with10% ammonia (15 ml) and extracted with ethyl acetate. Organic extractwas washed with saturated sodium chloride, dried over magnesium sulfateand evaporated. Column chromatography (5% MeOH-DCM) afforded 197 mg(35%) of the target compound as a yellowish solid. The ¹H NMR spectrawas consistent with the structure of Compound 22.

Example 9

Preparation of 1-phenyl-5-methyl-2-pyridinethione (Compound 18):1-phenyl-5-methyl-2-pyridinone (555.7 mg, 3 mmol) was reacted withLawesson's reagent (606.7 mg, 1.5 mmol) in toluene (5 ml) at 90° C.Reaction mixture was evaporated and the target compound was isolated bycolumn chromatography (20-30% ethyl acetate-hexane) followed bycrystallization from methyl-tert-butyl ether. Yield 403 mg (67%), yellowsolid. The ¹H NMR spectra was consistent with the structure of Compound18.

Example 10

Compound 33 was prepared according to the following synthetic scheme:

A mixture of commercially available ethyl 3,3-diethoxypropionate (9.7ml, 50 mmol), sodium hydroxide (10M, 6 ml, 60 mmol) and water (15 ml)was refluxed until homogenous (approximately 30 min). After cooling to0° C. 6N hydrochloric acid was added to bring pH of the solution to 2-3(˜10 ml). The mixture was extracted with dichloromethane, organic phasewas washed with water, dried over sodium sulfate, and the solvent wasremoved under vacuum to give acid 1 which was used without anyadditional purification.

To a solution of crude acid 1 (approximately 50 mmol) in DCM (100 ml) at0° C. was sequentially added 4-bromoaniline (10.3 g, 60 mmol), HOST (675mg, 5 mmol) and finally DCC (12.4 g, 60 mmol). The reaction mixture wasstirred at 0° C. for 1 hour and then refluxed for another 4 hours. Thesolid was filtered off, filtrate was washed with saturated sodiumbicarbonate, dried over sodium sulfate, and the solvent was removedunder vacuum to give amide 2 as slightly yellow solid. This solid wasdissolved in sulfuric acid (96%, 50 ml) at 0° C. The solution was keptat the same temperature for another 3 h and then poured into ice-water(500 ml). The solid was filtered off, washed with water, and stirredwith hot acetonitrile (100 ml). Quinolinone 3 was filtered off and driedunder vacuum. The yield was 10 g (91%).

Mixture of compound 3 (309 mg, 1.38 mmol), phenylboronic acid (336 mg,2.76 mmol), Cu(OAc)₂ (36 mg, 0.2 mmol), molecular sieves 4a (0.3 g),pyridine (0.24 ml) and DCM (10 ml) was stirred at room temperature for 2days. Reaction mixture was filtered trough Celite, washed with saturatedsodium bicarbonate with EDTA and organic phase was dried over sodiumsulfate. Compound 33 was isolated by chromatography (50%ethylacetate-hexane-ethylacetate). The yield was 352 mg (85%).

Example 11

The pharmacokinetic (PK) properties of pirfenidone, pirfenidone analogsand derivatives were assessed in dual canulated (right jugular/leftcarotid) Sprague Dawley rats (Charles River Laboratories, Inc.,Wilmington, Mass.). Male rats weighing approximately 275-300 g wereadministered an intravenous (5 mg/Kg) or oral (50 mg/Kg via gavage) doseof compound in an appropriate formulation. Plasma samples were collectedvia intra-arterial canula at desired times in the 24 hours after dosingusing EDTA as an anticoagulant. Three animals were used for eachcompound. All experiments were conducted by trained personnel inaccordance with guidelines of the appropriate Institutional Animal Careand Use Committees (IACUC).

Compound concentrations were assessed by LC-MS using a MDS SCIEX API3000 mass spectrometer (Applied Biosystems, Foster City, Calif.) coupledto a Shimadzu VP HPLC (Shimadzu Corp., Kyoto, Japan) outfitted with aDuragel G C₁₈ guard cartridge (Peeke Scientific, Redwood City, Calif.).Calibration samples were prepared by mixing known amounts ofpirfenidone, or a pirfenidone analog or derivative, with rat plasma. Astandard curve was created by serial dilution of the calibration samplein the same matrix. Both standard and analytical samples were preparedfor injection to the HPLC by mixing an aliquot of plasma sample with 3volumes of ice cold acetonitrile containing internal standard. Sampleswere then centrifuged. An aliquot of the resulting supernatant was thenmixed with five volumes of 0.2% formic acid in water, injected to theHPLC, and resolved in a methanol gradient (containing 0.18-0.2% formicacid). The integrated analyte signal was corrected for that of theinternal standard and compared to the appropriate standard curve inorder to define the analyte concentration.

The pharmacokinetic parameters shown in Table 4 were derived using theWinNonlin Software package (Pharsight Corp, Mountain View, Calif.).

TABLE 4 AUClast Route of Dose MRTinf Clobs ng-hr/mL × Admin. mg/kgCompound T_(1/2) hr. hr mL/hr/kg 10⁶ E % IV 5 8 13.9 20.3 0.117 30.2 192.96 6.26 0.142 35 26 1.62 2.4 0.691 10.3 Oral 50 8 122.4 40.5 19 191.754.8 26 65.8 63.9

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables,appendices, patents, patent applications and publications, referred toabove, are hereby incorporated by reference in their entirety.

1.-92. (canceled)
 93. A method of modulating a stress activated protein kinase (SAPK) system, comprising contacting a compound selected from the group consisting of

or a pharmaceutically acceptable salt or ester thereof; with a p38 mitogen-activated protein kinase (MAPK).
 94. A method of treating or preventing an inflammatory or fibrotic condition comprising administering to a subject in need thereof a effective amount of at least one compound selected from the group consisting of

or a pharmaceutically acceptable salt or ester thereof; wherein the effective amount produces a blood or serum or other bodily fluid concentration that is less than an EC₃₀ for inhibition for at least one p38 MAPK.
 95. The method of claim 94, wherein the anti-inflammatory or fibrotic condition is selected from the group consisting of fibrosis, chronic obstructive pulmonary disease, inflammatory pulmonary fibrosis, idiopathic pulmonary fibrosis, bronchiolitis obliterans syndrome, chronic allograft fibrosis, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gout, sepsis, septic shock; endotoxic shock, gram-negative sepsis, toxic shock syndrome, myofacial pain syndrome (MPS), Shigellosis, asthma, adult respiratory distress syndrome, inflammatory bowel disease, Crohn's disease, psoriasis, eczema, ulcerative colitis, glomerular nephritis, scleroderma, chronic thyroiditis, Grave's disease, Ormond's disease, autoimmune gastritis, myasthenia gravis, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, pancreatic fibrosis, chronic active hepatitis, hepatic fibrosis, renal disease, renal fibrosis, irritable bowel syndrome, pyresis, restenosis, cerebral malaria, stroke and ischemic injury, neural trauma, Alzheimer's disease, Huntington's disease, Parkinson's disease, acute or chronic pain, an allergy, cardiac hypertrophy, chronic heart failure, acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease, Reiter's syndrome, acute synoviitis, muscle degeneration, bursitis, tendonitis, tenosynoviitis, herniated, ruptured, or prolapsed intervertebral disk syndrome, osteopetrosis, thrombosis, silicosis, pulmonary sarcosis, bone resorption disease, cancer, Multiple Sclerosis, lupus, fibromyalgia, AIDS, Herpes Zoster, Herpes Simplex, influenza virus, Severe Acute Respiratory Syndrome (SARS), cytomegalovirus, and diabetes mellitus.
 96. The method of claim 94, wherein the effective amount is less than 50% of an amount that causes an undesirable side effect in the subject.
 97. The method of claim 94, wherein the compound inhibits a kinase in the SAPK signaling pathway.
 98. The method of claim 94, wherein the administering of the compound is on a schedule selected from the group consisting of twice a day, once a day, once every two days, three times a week, twice a week, and once a week.
 99. The method of claim 95, wherein the administering of the compound is on a schedule selected from the group consisting of twice a day, once a day, once every two days, three times a week, twice a week, and once a week.
 100. The method of claim 95, wherein the anti-inflammatory or fibrotic condition is bronchiolitis.
 101. The method of claim 95, wherein the anti-inflammatory or fibrotic condition is chronic allograft fibrosis.
 102. The method of claim 95, wherein the anti-inflammatory or fibrotic condition is idiopathic pulmonary fibrosis.
 103. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 104. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 105. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 106. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 107. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 108. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 109. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 110. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 111. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 112. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 113. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 114. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 115. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 116. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 117. The method of claim 94, wherein the compound has a structure

or a pharmaceutically acceptable salt or ester thereof.
 118. A method of identifying a pharmaceutically active compound, comprising: assaying a plurality of compounds from a library of compounds for inhibition of at least one p38 MAPK; and selecting at least one compound from the plurality of compounds, wherein the selected compound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μM for inhibition of the at least one p38 MAPK.
 119. A method of identifying a pharmaceutically active compound, comprising: assaying a plurality of compounds from a library of compounds for inhibition of TNFα secretion in a bodily fluid in vivo; and selecting at least one compound from the plurality of compounds, wherein the selected compound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μM for inhibition of TNFα secretion in a bodily fluid in vivo.
 120. A compound having the formula of Subgenus III:

wherein X₃ is selected from the group consisting of H, F, and OH; R₂ is selected from the group consisting of H and CF₃; and wherein the compound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μM for inhibition of p38 MAPK; or a pharmaceutically acceptable salt, ester, solvate or prodrug of the compound.
 121. A compound having the formula of Genus VII:

wherein X₃ is H, halogen, alkoxy, or OH; Y₁, Y₂, Y₃, and Y₄ are independently selected from the group consisting of H, C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₁-C₁₀ alkenyl, C₁-C₁₀ haloalkyl, C₁-C₁₀ nitroalkyl, C₁-C₁₀ thioalkyl, C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ alkoxy, phenyl, substituted phenyl, halogen, hydroxyl, C₁-C₁₀ alkoxyalkyl, C₁-C₁₀ carboxy, C₁-C₁₀ alkoxycarbonyl; R₄ is H, halogen, or OH; and wherein the compound exhibits an EC₅₀ in the range of about 1 μM to about 1000 μM for inhibition of p38 MAPK; or a pharmaceutically acceptable salt, ester, solvate or prodrug of the compound. 